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You are an expert at summarizing long articles. Proceed to summarize the following text: its water is used for domestic , municipal , industrial , agricultural , and recreational purposes . besides , the tigris is thought to be the ultimate sink for all wastewater arising from above activities . many papers and theses were published regarding its sources of pollution , pollutants concentration , degradation in water quality , and other aspects [ 15 ] . this paper is another effort added to previous works trying to shed light on evaluating status of the river as it passes through a selected stretch within mosul city . such stretch includes the major point and non point sources of pollution that do affect river water quality and quantity . a detailed survey was made to figure out the activities that might contribute to the pollution of the tigris within mosul city ( fig 1 ) . physical , chemical , and biological tests had been conducted on each adopting the standard methods ( 6 , 7 ) . the tigris - like any other water resource - is subjected to numerous sources of pollution . municipal discharges , industrial pollutants , agricultural activities residuals , direct runoff , tourism , illegal practices , atmospheric pollution , and others are few examples . the most important sources covered for the purpose of this paper are listed in table ( 2 ) . it is estimated that as huge as 400000 m of wastewater is daily discharged - untreated - into the river . this is equivalent to 17000 m /hr with a peak of 20000m / hr at day hours . domestic waste loads , on the one hand , add a great burden on the pollution of the tigris . these wastes comprise foul wastes of more than 6000 dwellings and apartments lacking waste collection system . considerable amounts of these wastes are directly or indirectly ( via valleys ) discharge their loads into the river ( table 3 ) the field survey reveals that some of the pollution sources are direct point sources while some other sources are indirectly affecting the river water quality . as these sources differ , the nature of their pollution loads vary accordingly as shown in tables ( 46 ) . the characteristics of discharged wastewater can be grouped into the medium - strong categories according to guidelines . concentration of various contaminants & compounds exceed the limits recommended by local and authorized agencies [ 9 , 10 ] . odor , foam , color , death and migration of aquatic life , and dominance of anaerobic conditions can easily be detected near sewer outfalls . these adverse consequences as well as the increase of pollutant concentrations have lessen the river aesthetics , increase hardness , salinity , and rendering the water unfit for different beneficial uses . the changes in the physical characteristics of the river water such as temperature , turbidity , and suspended solids are clearly demonstrated in table 7 . these changes are detected as the river passes along the stretch of the study area . in the north of the city animal breeding ( buffalo , sheep and cows ) are widespread . in the heart of the city the total bacterial count in the discharged wastewater is amounted as high as 210 210 . this disorder is attributed to some illegal practices such as discharging toilet waste directly into the river or due to in - house slaughtering activities . eutrophication , a phenomenon that largely takes place at lakes and slow moving water bodies has found an access to occur in the tigris . this problem arises from the fact of using large amounts of nutrient - rich detergents . throughout the past years , detergents were distributed to the families as a part of a monthly ration of oil for food program during sanction ( 1991-present ) . such detergents contained high concentrations of phosphorous the main cause of eutrophication , ( see table 8) . moreover , eutrophication can be detected by chlorophyll measurement which surpasses the guidelines of 2mg / l . eutrophication is known of its vast adverse effects , some of which are listed in table ( 9 ) . the bod5 values represent the organic pollution of the tigris . organic load leads to decline of dissolved oxygen and release of ammonia and nitrite . this table indicates that river water can be classified as poor - good in terms of quality as per authorized standards . it does show that the tigris starts recovering its health after 40 km . on the other hand , the survey revealed the huge adverse impacts incurred by industry , tourism and health - care institutions . moreover , such plants are secondary and incapable of removing nutrients ( p&n compounds ) as well as they poorly perform at shock loads occasions . heavy metals such as chrome , copper , and arsenic may have an access to reach the river . some industries produce these elements in their processes like textiles industries , tanneries , etc . al layla & al - rawi confirm this fact upon studying impact of textile wastewater discharges on the tigris . the problem with heavy metal is that they are absorbed by particulates at normal ph levels causing very low dissolved traces and consequently its monitoring becomes very complicated . this is reflected on an increase in organic & bacterial loads , and causing health risks . industrial , tourism , medical institutions and other services add to the pollution of the river impeding self purification and rendering the water unfit for different uses . the tigris starts recovering health and resumes an acceptable quality after 40 km from reference point . absence of awareness of the pollution prevention measures and the non - existing of a real environmental monitoring authority increased pollution of the tigris . water and sewerage directorates should play an effective role in preventing unlawful connections to water networks and reducing hydraulic loads received by the river . environmental authority should be given the power to enforce the law against illegal practices . all types of media should contribute to raise the public awareness of environmental protection as a holy and human task recommended by all religions ( islam , christianity , etc ) .
this paper presents an overall view of major sources that may lead to the pollution of the tigris within mosul city . a stretch exceeding 20kms in length is selected that represents the sick path of the river . many sites along the studied stretch are likely to affect the river quality in some way or another . samples from 40 sources sites are taken for quality analyses these sources as huge as 400000 m3 a day are characterized as ( medium strong ) in composition . such wastewaters with the pollutants they carry alter the river water quality rendering it unsuitable for beneficial uses . such alterations do leave many negative consequences concerning human beings and aquatic life . it is found that domestic discharges are among the most important sources of pollution . sanitary wastes are often discharged untreated -into the tigris . other illegal practices such as in - house slaughtering add to the pollution as well . industrial , tourist and institutional wastes put an additional burden on pollution of the river water quality . these wastes contain lead , chrome , and other heavy metals that may pose health risks . wastewater treatment plants that exist in some sectors do not perform as they are expected . they need proper evaluation and rehabilitation . eutrophication - a characteristic problem in lakes - finds an access to occur into the tigris . this problem results from intensive use of detergents rich in nutrients ( p&n compounds ) . in general , pollutants of different sources heavily affect the river water . recovery and self purification of the river is estimated to occur at 40 km far from reference point . the paper concludes with the necessity of construction of a central treatment plant(s ) or tackling the pollutants at their origin . the paper also stresses on importance of environmental education and awareness in order to combat pollution problems .
You are an expert at summarizing long articles. Proceed to summarize the following text: in new york , need for pep is determined by outcome of 10-day confinement ( of all domestic animals ) or laboratory testing ( all species ) . healthcare providers report suspected rabies exposures to local health departments , which absorb authorized pep costs beyond those borne by third - party payers and partial reimbursement by the new york state department of health ( 9 ) . exposures to bats and humans , animals submitted only for surveillance , and data from new york city ( not part of the reporting system ) were excluded . we analyzed data with sas version 9.2 ( sas institute , cary , nc , usa ) using us census data for rates ( www.factfinder.census.gov ) . because of skewed distributions , we used spearman rank correlation coefficients for measures of association . the number of terrestrial animals submitted declined 56% from 10,552 in 1993 to 4,631 in 2002 . the number and proportion of rabid animals , which decreased from 2,637 ( 25.0% ) in 1993 to 608 ( 13.1% ) in 2002 , were strongly associated with the number of submitted animals ( spearman r = 0.99 , p<0.0001 ) . for 70.4% of the 13,004 exposure incidents during 19932002 , untestable and positive animals accounted for 2.6% and 23.4% of pep , respectively . for 3.6% of exposure incidents , pep began before rabies was ruled out . * each rabies exposure situation in which > 1 persons underwent pep was defined as an incident . exposure incidents declined 45% , from 1,815 in 1993 to 1,006 in 2001 ( figure 1 ) . pep decreased from 2,755 ( 25.3 peps/100,000 persons ) in 1993 to 1,327 in 2000 ( 12.1 pep/100,000 persons ) . each year , the number of persons receiving pep correlated with the number of submitted animals ( spearman r = 0.94 , p<0.0001 ) and rabid animals ( spearman r = 0.95 , p<0.0001 ) . although fewer cats ( 303 ) than raccoons ( 8,318 ) were rabid , cats accounted for the most exposure incidents ( 4,266 [ 32.8% ] ) and pep ( 5,777 [ 31.8% ] ) ( table 2 ) . dogs accounted for 3,052 ( 23.5% ) exposure incidents and 3,435 ( 18.9% ) pep . in new york , dogs and cats accounted for a high proportion of pep from animals without rabies determination ( 85.3% and 67.6% , respectively ) . raccoons accounted for 3,298 ( 25.4% ) exposure incidents and for 5,210 ( 28.7% ) pep . from 1993 to 2002 , the proportion of pep attributed to raccoons changed from 48% to 22% ; cats , from 21% to 35% ; and dogs , from 11% to 22% . terrestrial rabies associated exposure incidents and postexposure prophylaxis ( pep ) use , by year , new york ( excluding new york city ) , usa , 19932002 . * each rabies exposure situation in which > 1 persons underwent pep was defined as an incident . scratches , saliva / nervous system tissue exposure , mucous membrane exposure , indirect exposure , or unknown . in 43 new york counties with populations < 200,000 , the pep rate averaged 33.7/100,000 ( range 8.481.3/100,000 ) . the 14 larger counties ( populations > 200,000 ) had significantly lower pep rates ( 9.8/100,000 , range 0.521.8/100,000 ; p<0.0001 ) and pep per exposure incident ( p<0.0001 ) but accounted for 42.6% of pep . during 19982002 when sex and age of exposed persons were reported , data were missing for 211 of 7,221 pep reports . persons who received pep did not differ by sex ( 3,625 male , 3,569 female ) . pep rates were highest for children 1014 years of age ( figure 2 ) . for male patients , pep rates were lower in older age groups ; for female patients , rates were highest in the 4044-year group . female patients received pep significantly more often because of cat exposures than did male patients ( 1,736 vs. 1,053 ; p<0.0001 ) . male patients received pep significantly more often from dog ( 984 vs. 583 ; p = 0.0005 ) and raccoon ( 767 vs. 595 ; p = 0.05 ) exposures than did female patients . for each age group , except the > 85-year age group , female patients received pep more often from cat exposures and male patients more often from dog exposures . rate of postexposure prophylaxis ( pep ) use per 100,000 persons per year , by sex and 5-year age groups , new york ( excluding new york city ) , 19982002 . the 8,405 bites accounted for 46.3% of pep . a total of 1,114 ( 6.1% ) of pep occurrences were associated with scratch exposures and 3,707 ( 20.4% ) with saliva / nervous tissue exposures . for indirect or unknown types of contact , 4,298 ( 27.2% ) pep for direct contact significantly exceeded that for indirect or unknown contact for the study period ( p<0.0001 ) and for each year except 1993 . bites accounted for significantly more pep because of dog and cat exposures ( 86.4% vs. 63.3% ; p<0.0001 ) than did scratches or saliva / nervous tissue exposures . raccoon exposures more frequently resulted from saliva / nervous tissue exposure than from bites ( 22.4% vs. 13.0% ; p<0.0001 ) . of 7,221 pep occurrences during 19982002 when local health department authorization was reported , 6,846 ( 94.8% ) were reported as authorized . of 6,264 persons not reported as previously vaccinated , 5,574 ( 89.0% ) received 5 vaccine doses and 5,563 ( 88.8% ) received human rabies immune globulin . pep completion was not reported ( no report received ) for 716 ( 11% ) persons ; 701 had no prior treatment history . most ( 79% ) incomplete pep in new york was associated with animals not captured for rabies determination . of 119 pep associated with rabies - negative animals , 108 ( 91% ) were not completed . pep were not started for 17 ( 1% ) and were not completed for 34 ( 2% ) of the 2,217 pep associated with rabid animals . most ( 697 [ 97% ] ) incomplete pep was from direct contact exposures , primarily bites ( 87% ) . a total of 33 ( 9% ) of 376 persons with adverse reactions did not complete treatment . incomplete pep was associated more often with exposures to dogs ( 42% ) and cats ( 42% ) than to other species . the rate in new york was lower than that in massachusetts when its epizootic was well established in 1995 ( 10 ) , perhaps because new york requires treating physicians to consult with local public health authorities . similar to rates in new york , pep rates in ontario , canada , decreased as fox rabies became enzootic and were weakly but significantly associated with animal rabies ( 11 ) . this association may be due to epizootic - related reductions in animal populations , resulting in fewer rabid animals and human contacts . unlike new york , in kentucky pep occurred more frequently after exposures to dogs than cats ( 12 ) . in kentucky , the proportion of incomplete pep was the same as in new york ( michael auslander , pers . treatment completion rates for new york and kentucky were higher than those in a study of 11 us emergency departments ( 65% ) ( 13 ) . in florida , 22% of pep were inappropriate according to a state algorithm ( 14 ) ; in new york , local health departments report few unauthorized pep administrations . in new york , over time and with education , pep associated with indirect exposures apparently can be reduced . of most concern more efforts are needed to capture exposing animals to rule out both rabies and the need for pep . capturing exposing animals should be a major component of animal control efforts that along with vaccination have been successful at reducing rabies risks .
during 19932002 , cats accounted for 2.7% of rabid terrestrial animals in new york but for one third of human exposure incidents and treatments . nonbite exposures and animals of undetermined rabies status accounted for 54% and 56% , respectively , of persons receiving rabies treatments .
You are an expert at summarizing long articles. Proceed to summarize the following text: transcriptional regulation of eukaryotic genes has classically been viewed as the interaction of elements in the immediate vicinity of the transcription start site ( promoters ) with upstream elements ( enhancers ) . however , transcriptional regulation is not only determined by dna sequence but involves additional layers of control that include nucleosome positioning , dna binding regulatory proteins such as transcription factors , histone modifications and non - coding rna [ 5 - 7 ] . moreover , the development of high throughput chromosome conformation capture techniques has shown that the three - dimensional organization of genes and upstream elements affects transcriptional activity [ 9 - 11 ] . here , we present how the combination of these factors can provide a context for the regulation of gene expression . gene expression regulation in eukaryotes includes interactions between promoters and enhancers , but our understanding of the mechanisms that drive these interactions , or that determine their specificity , is far from complete . at the basis of transcription lies the dna code that directly determines the composition and location of dna elements and provides specific recognition sites for dna binding proteins . the binding of transcription factors and recruitment of complexes that modify histones create an environment that allows for element interaction and initiation of gene transcription . however , predicting the location of promoters and enhancers based solely on histone modifications and transcription factor binding relies on complicated models that are still suboptimal [ 12 - 14 ] . moreover , recent findings on the involvement of non - coding rna in transcriptional regulation further imply a more complicated reality . is there a combination of histone modifications which is sufficient to predict the position of regulatory elements ? it is now highly debated whether chromatin modifications comprise a code similar to dna ( reviewed by henikoff and shilatifard ) . the crosstalk between histones , transcription factors and non - coding rna suggest that they interact to form a highly interwoven level of organization . in the context model , transcriptional regulation is subdivided into three levels of interactions : the dna level , the local chromatin level , and the three - dimensional folding of the genome . the first level , the dna code , forms an interaction platform by providing protein binding sites for transcription factors that , together with non - coding rnas and histone modifications , form the next layer of gene regulation . this layer of interactions enables specific long - range interactions that result in a three - dimensional folding of the chromatin . this higher order organization provides transcriptional context that can either facilitate or block the initiation of transcription ( figure 1 ) . compaction of higher order structures , for example , will influence the accessibility of dna and binding of transcription factors , providing a likely feedback mechanism . ( a ) the context model of gene expression consists of three layers of regulation , each representing a level of interactions that allows the transition from code to context . ( b ) dna , the first layer , interacts with non - coding rna ( arbitrary sequence in blue ) , tfs ( green ) and histone modifications ( red ) . the third layer is the resulting 3d folding ( dotted line ) , which creates the final context for gene transcription . abbreviations : ncrna , non - coding rna ; tf , transcription factor ; 3d , three - dimensional . the binding of ( core ) transcription factors critically depends on the recognition of specific dna sequences , known as dna motifs . high throughput techniques , such as chip - seq and enhanced yeast one - hybrid , which visualizes the interaction of a transcription factor with a bait dna sequence , are now employed to uncover transcription factor - dna and dna - transcription factor interactions , respectively . such studies show that transcription factor - binding events are abundant and can occur at large genomic distances from genes . they may affect the binding affinity of factors , or encode non - coding rnas . interestingly , deletion of these sequences with unknown function can influence gene expression of genes located hundreds of kb away , implying that long - range looping of dna brings the sequences into contact with the genes they regulate . this level of interactions involves histone positioning and modifications , repertoires of transcription factors and non - coding rna and the interplay between them . this level is perhaps the most complex , and it has become the center of attention in recent years . although nucleosome positioning , histone modifications , non - coding rna and transcription factor binding are useful descriptors for genomic elements , they do not seem to define regulatory elements by themselves . histone modifications have been used to classify upstream regions as promoters ( i.e. h3k4me3 ) and enhancers ( i.e. h3k4me1 , h3k27ac ) , but this may not reflect the complete picture . recent developments in dna sequencing have led to the appreciation of the importance of non - coding rna as a regulatory component in the genome . although the extent of non - coding rna involvement is still debated , more and more examples of the involvement of non - coding rna in transcriptional regulation appear in the current literature . their function is still mostly unknown , and their nomenclature is purely descriptive , based on their site of occurrence ( e.g. pasr for promoter - associated short rna or erna for enhancer - rna ) . it is possible that non - coding rna can act as a fast and flexible intermediate to recruit histone - modifying complexes to dna elements [ 29 - 32 ] . several reports on long non - coding rna have shown their involvement in chromatin remodeling , affecting differentiation and disease ( reviewed by huarte and rinn and hung and chang ) . long non - coding rnas have also been found to be involved in transcriptional repression via polycomb proteins , which are known to maintain cell identity by repressing developmental regulators in certain cell types . although long non - coding rna are now widely studied , small non - coding rna and antisense rna have also been implicated in polycomb - mediated transcriptional gene silencing [ 36 - 38 ] . the combinatorial complexity at this level of chromatin regulation and structure is further modulated by feedback and feedforward signals between histone modifications , non - coding rna and transcription factors . the three - dimensional folding of dna is the final context that allows for gene transcription to initiate . the folding of dna into higher order structures is not a random event , and it has long been thought to affect gene transcription [ 39 - 42 ] . at the nuclear level , chromosomes occupy specific nuclear territories ( reviewed by cremer and cremer ) . chromatin interactions within and between broad zones of chromosomes lead to nuclear compartments where active genes tend to co - locate , near the center of the nucleus , and inactive genes cluster near the nuclear periphery . this indicates a strong correlation , though not necessarily causation , between nuclear positioning and gene activity ( reviewed by geyer et al . ) . at the finest scale , precise dna folding or dna looping ( level 3 ) is guided by long - range interactions between dna sequence elements ( level 1 ) , which can be mediated by interacting context creators ( level 2 ) . an illustration of the interplay between multiple levels of regulation that leads to a context for gene expression can be found in the regulation of the hoxa locus . here , chromosomal looping brings the non - coding rna hottip in close proximity to hoxa genes . hottip recruits the histone 3 lysine 4 modifying complex mll by binding to wdr5 , targeting this complex to the hoxa locus . as a result , hottip controls hoxa gene expression by bridging higher - order chromosomal looping and chromatin modifications . this exemplifies how context provides an environment for communication between regulatory elements in three - dimensional space , leading to either activation or repression of gene transcription . gene expression regulation in eukaryotes includes interactions between promoters and enhancers , but our understanding of the mechanisms that drive these interactions , or that determine their specificity , is far from complete . at the basis of transcription lies the dna code that directly determines the composition and location of dna elements and provides specific recognition sites for dna binding proteins . the binding of transcription factors and recruitment of complexes that modify histones create an environment that allows for element interaction and initiation of gene transcription . however , predicting the location of promoters and enhancers based solely on histone modifications and transcription factor binding relies on complicated models that are still suboptimal [ 12 - 14 ] . moreover , recent findings on the involvement of non - coding rna in transcriptional regulation further imply a more complicated reality . is there a combination of histone modifications which is sufficient to predict the position of regulatory elements ? it is now highly debated whether chromatin modifications comprise a code similar to dna ( reviewed by henikoff and shilatifard ) . the crosstalk between histones , transcription factors and non - coding rna suggest that they interact to form a highly interwoven level of organization . in the context model , transcriptional regulation is subdivided into three levels of interactions : the dna level , the local chromatin level , and the three - dimensional folding of the genome . the first level , the dna code , forms an interaction platform by providing protein binding sites for transcription factors that , together with non - coding rnas and histone modifications , form the next layer of gene regulation . this layer of interactions enables specific long - range interactions that result in a three - dimensional folding of the chromatin . this higher order organization provides transcriptional context that can either facilitate or block the initiation of transcription ( figure 1 ) . compaction of higher order structures , for example , will influence the accessibility of dna and binding of transcription factors , providing a likely feedback mechanism . ( a ) the context model of gene expression consists of three layers of regulation , each representing a level of interactions that allows the transition from code to context . ( b ) dna , the first layer , interacts with non - coding rna ( arbitrary sequence in blue ) , tfs ( green ) and histone modifications ( red ) . the third layer is the resulting 3d folding ( dotted line ) , which creates the final context for gene transcription . abbreviations : ncrna , non - coding rna ; tf , transcription factor ; 3d , three - dimensional . the binding of ( core ) transcription factors critically depends on the recognition of specific dna sequences , known as dna motifs . high throughput techniques , such as chip - seq and enhanced yeast one - hybrid , which visualizes the interaction of a transcription factor with a bait dna sequence , are now employed to uncover transcription factor - dna and dna - transcription factor interactions , respectively . such studies show that transcription factor - binding events are abundant and can occur at large genomic distances from genes . they may affect the binding affinity of factors , or encode non - coding rnas . interestingly , deletion of these sequences with unknown function can influence gene expression of genes located hundreds of kb away , implying that long - range looping of dna brings the sequences into contact with the genes they regulate . this level of interactions involves histone positioning and modifications , repertoires of transcription factors and non - coding rna and the interplay between them . this level is perhaps the most complex , and it has become the center of attention in recent years . although nucleosome positioning , histone modifications , non - coding rna and transcription factor binding are useful descriptors for genomic elements , they do not seem to define regulatory elements by themselves . histone modifications have been used to classify upstream regions as promoters ( i.e. h3k4me3 ) and enhancers ( i.e. h3k4me1 , h3k27ac ) , but this may not reflect the complete picture . recent developments in dna sequencing have led to the appreciation of the importance of non - coding rna as a regulatory component in the genome . although the extent of non - coding rna involvement is still debated , more and more examples of the involvement of non - coding rna in transcriptional regulation appear in the current literature . their function is still mostly unknown , and their nomenclature is purely descriptive , based on their site of occurrence ( e.g. pasr for promoter - associated short rna or erna for enhancer - rna ) . it is possible that non - coding rna can act as a fast and flexible intermediate to recruit histone - modifying complexes to dna elements [ 29 - 32 ] . several reports on long non - coding rna have shown their involvement in chromatin remodeling , affecting differentiation and disease ( reviewed by huarte and rinn and hung and chang ) . long non - coding rnas have also been found to be involved in transcriptional repression via polycomb proteins , which are known to maintain cell identity by repressing developmental regulators in certain cell types . although long non - coding rna are now widely studied , small non - coding rna and antisense rna have also been implicated in polycomb - mediated transcriptional gene silencing [ 36 - 38 ] . the combinatorial complexity at this level of chromatin regulation and structure is further modulated by feedback and feedforward signals between histone modifications , non - coding rna and transcription factors . the three - dimensional folding of dna is the final context that allows for gene transcription to initiate . the folding of dna into higher order structures is not a random event , and it has long been thought to affect gene transcription [ 39 - 42 ] . at the nuclear level , chromosomes occupy specific nuclear territories ( reviewed by cremer and cremer ) . chromatin interactions within and between broad zones of chromosomes lead to nuclear compartments where active genes tend to co - locate , near the center of the nucleus , and inactive genes cluster near the nuclear periphery . this indicates a strong correlation , though not necessarily causation , between nuclear positioning and gene activity ( reviewed by geyer et al . ) . at the finest scale , precise dna folding or looping interactions between gene promoters and their distal regulatory elements can be found . dna looping ( level 3 ) is guided by long - range interactions between dna sequence elements ( level 1 ) , which can be mediated by interacting context creators ( level 2 ) . an illustration of the interplay between multiple levels of regulation that leads to a context for gene expression can be found in the regulation of the hoxa locus . here , chromosomal looping brings the non - coding rna hottip in close proximity to hoxa genes . hottip recruits the histone 3 lysine 4 modifying complex mll by binding to wdr5 , targeting this complex to the hoxa locus . as a result , hottip controls hoxa gene expression by bridging higher - order chromosomal looping and chromatin modifications . this exemplifies how context provides an environment for communication between regulatory elements in three - dimensional space , leading to either activation or repression of gene transcription . recent technical advances in dna sequencing have enabled genome - wide analysis at each of the three levels : ( 1 ) genome sequencing to identify conserved regulatory elements ; ( 2 ) chip - seq , deepcage , rna - seq , net - seq , chirp and chart to identify chromatin modification , transcription factor binding and ( non - coding ) rna expression and localization ; and ( 3 ) 3c combined with deep sequencing ( e.g. hi - c and 3-seq ) to probe three - dimensional folding of the genome . combined , these tools allow an integrated systems approach towards a more complete understanding of the context in which the genome is regulated .
recent advances in sequencing technologies have uncovered a world of rnas that do not code for proteins , known as non - protein coding rnas , that play important roles in gene regulation . along with histone modifications and transcription factors , non - coding rna is part of a layer of transcriptional control on top of the dna code . this layer of components and their interactions specifically enables ( or disables ) the modulation of three - dimensional folding of chromatin to create a context for transcriptional regulation that underlies cell - specific transcription . in this perspective , we propose a structural and functional hierarchy , in which the dna code , proteins and non - coding rnas act as context creators to fold chromosomes and regulate genes .
You are an expert at summarizing long articles. Proceed to summarize the following text: in many calcium - regulated exocytotic systems , the response to increasing concentrations of intracellular calcium is characterized by a sigmoidal relationship between exocytosis and calcium concentration ( reviewed in knight and scrutton , 1986 ; knight and baker , 1987 ) . there exist calcium concentrations that can elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse , despite prolonged exposure to calcium concentrations exceeding a threshold value . since exocytosis is an irreversible secretion of concentrated products , submaximal exocytotic responses can not be explained with single , linear reaction schemes because the irreversible step will collect , in time , the entire population . we have examined the origin of submaximal exocytotic responses because ( a ) differentiation between hypotheses that attempt to explain this behavior is required for the development of correct secretory models ; ( b ) it is a feature common to calcium - triggered exocytotic systems , including synaptic transmission and neuroendocrine secretion ; and ( c ) the goal of relating specific proteins with steps in the exocytotic process can only proceed if the steps in the process are correctly identified and characterized in detail . in the sea urchin , strongylocentrotus purpuratus , 50% of the vesicles fuse in response to 20 m free calcium ( vogel et al . , 1996 ) . in this paper , we consider two general hypotheses to explain why only a fraction of the vesicles fused . the first hypothesis is that an initially homogeneous population of cortical vesicles is inactivated after the initiation of the fusion process such that only a fraction fuse . the second hypothesis is that the population of cortical vesicles is heterogeneous in their response to calcium before the application of calcium , such that different subpopulations of vesicles enter the fusion process , depending upon the magnitude of the calcium stimulus . models have been proposed for synaptic transmission and neuroendocrine secretion that attempt to quantitatively relate the extent and/or rate of exocytosis to the calcium concentration ( dodge and rahamimoff , 1967 ; llinas et al . , 1976 , 1981 ; simon and llinas , 1985 ; zucker and fogelson , 1986 ; heinemann et al . , 1993 , 1994 ) . modeling the exocytotic response often requires the inclusion of calcium - dependent steps distinct from the actual fusion step . these steps may be dependent upon temporal and spatial properties of the calcium concentration that obscure the underlying relationship between calcium and exocytosis . preparations that reduce the degree of complexity are attractive systems for investigating the relationship between calcium and exocytosis . cortical degranulation in the sea urchin egg isolated planar cortex , an example of calcium - triggered exocytosis , is such a preparation ( vacquier , 1975 ; baker and whitaker , 1978 ; zimmerberg et al . , 1985 ; reviewed in jackson and crabb , 1988 ; vogel et al . it is ideal for these studies because the exocytotic response to calcium is sigmoidal , vesicles are pre - docked , endocytosis does not occur , and calcium - triggered exocytosis occurs in the absence of atp , gtp , and all other soluble factors and proteins . vesicle exocytosis is neither influenced by nor dependent upon interconversion between different spatially localized pools or compartments . in other systems , complications such as the temporal overlap of exocytosis and endocytosis , or the loss of cytosolic factors , rundown , make the origin of submaximal responses difficult to evaluate . for example , the interpretation of membrane capacitance changes during exocytosis is strongly dependent upon differentiation of vesicle transport between reserve and readily releasable compartments and from endocytosis ( heinemann et al . , 1994 ) , and manipulating the submaximal state in certain permeabilized cell preparations is limited by the loss of factors required for exocytosis ( sarafian et al . , 1987 ) . no evidence was found to support the hypothesis that calcium - dependent inactivation is responsible for the cessation of fusion ( submaximal responses ) . the hypothesis that the calcium sensitivity of secretory vesicle exocytosis in vitro is heterogeneous before initiating fusion was consistent with the data . a decrease in the number of vesicles within a fusing subpopulation was the only limiting process identified . specimens of the sea urchins , strongylocentrotus purpuratus and lytechinus pictus were purchased from marinus , inc . ( long beach , ca ) and maintained at 10 and 15c in aquaria , respectively . eggs were obtained by intracoelomic injection of 0.5 m kcl and collected in artificial sea water ( instant ocean ; aquarium systems , mentor , oh ) . eggs were dejellied mechanically by passage through a 90 ( s. purpuratus ) or 110 ( l. pictus ) m pore size nylon mesh , washed three times in large volumes of artificial sea water , allowed to settle in a large beaker that minimized packing , and held at 16c in artificial sea water . an aliquot of the egg suspension was deposited onto poly - l - lysine - treated ( 250 g / ml ) cover slips and the eggs were allowed to settle and attach to the coverslip over a 25-min period . the cover slip was rinsed with shearing medium : 50 mm piperazine - ethane sulfonic acid , k - pipes ( sigma chemical co. , st . louis , mo ) , 425 mm potassium chloride ( mallinckrodt , inc . , paris , ky ) , 3 mm hydroxyethyl - ethylenediamine - triacetic acid ( hedta ; sigma chemical co. ) , 3 mm egta ( fluka chemical corp . , ronkonkoma , ny ) , 3 mm nitrilotriacetic acid ( nta ; fluka chemical corp . ) , and 1 mm benzamidine ( sigma chemical co. ) , ph 6.7 adjusted with potassium hydroxide ( mallinckrodt , inc . ) . later experiments used a modified calcium buffer containing 6 mm hedta , 6 mm egta , and 3 mm nta . the exocytotic response to defined calcium concentrations was identical using either buffer composition . a stream of 25 ml of shearing medium produced from a hand - held syringe with a 19-gauge needle was used to shear the attached eggs . this procedure resulted in an attached layer of plasma membrane with bound cortical vesicles ( vacquier , 1975 ) , known as the planar isolated cortex . solutions with defined calcium concentrations were prepared by adding cacl2 ( 0.1000 0.0005 mol / liter calcium standard ; orion research , cambridge , ma ) to shearing medium ( 07 mm final concentration ) . calcium activities were measured at room temperature ( 2124c ) with calcium - specific electrodes ( tipca , kwik tip ; world precision instruments , inc . , solutions prepared according to tsien and rink ( 1980 ) , and commercial solutions ( calbuf-1 ; world precision instruments , inc . ) gave the same calibration curves . the voltage response of the calcium electrodes was linear over the pca range 7.03.0 . the standard error for any single pca determination was typically < 0.1 pca u. the standard error for averaged data was always < 0.1 pca u and was typically < 0.05 pca u. fast perfusion of solutions was achieved using a flow chamber specifically designed for use with multiple solution exchange protocols ( kaplan et al . , 1991 , 1996 ; zimmerberg et al . , 1992 ) . the original perfusion system was modified for use with four solutions instead of two . four valves were used for solution delivery , two for solution removal , and two were used to toggle the vacuum lines for rapid exchange of fluid . unstirred layers were minimized using flow rates fast enough to generate turbulent flow within the chamber . solution exchange at the cover slip surface was estimated to be < 200 ms based on the ph - dependent shift in the fluorescence of coverslips coated with fluorescein - conjugated albumin ( kaplan et al . , 1996 ) . later experiments used a modified perfusion system using unidirectional solution exchange within the flow chamber . the scattered intensity is proportional to the number of cortical vesicles present in the monolayer of cortices : the scattered intensity decreases as the number of cortical vesicles decreases ( zimmerberg et al . , 1985 ) . an upright microscope fitted with a photodiode detector was used as the light scattering photometer . the combination of a darkfield condenser ( 0.63 numerical aperture ) and a low numerical aperture objective ( 0.22 numerical aperture ) provided conical collection over scattering angles ranging from 1040 degrees . light scattering was normalized using the initial and final intensities measured in the absence of calcium and after exposure to a high calcium concentration ( > 300 m ) . . exposure of the cortices to high calcium concentrations resulted in the fusion of essentially every cortical vesicle present . the probed monolayer consisted of 2040 cortices , resulting in a signal that represented the scattering behavior of 10 cortical vesicles . differences in the free calcium concentration of solutions measured at room temperature but used at 16c were estimated using the program maxchelator ( v6.95 , bers constants ; program provided by chris patton , [email protected] ) and found to be less than the standard error for pca determination . the data obtained from 188 egg preparations ( 111 s. purpuratus and 77 l. pictus ) over a 3-yr period are presented in this study . the results from a total of 94 double challenges ( 68 s. purpuratus and 26 l. pictus ) are reported . curve fitting was performed using the levenburg - marquardt algorithm with pearson minimization as implemented in tablecurve 2d software ( windows v2.0 ; jandel scientific , corte madera , ca ) . derivative curves were calculated from percent fusion versus time using third - order forward differences and smoothed using a low - pass filter as implemented in sigmaplot ( windows v2.0 ) . no seasonal or yearly differences in exocytosis were observed except at the end of the season , when large decreases in calcium sensitivity and loss of resistance to shear were sometimes observed . decreases in the calcium sensitivity of the cortex during the first few minutes after shearing have been reported ( moy et al . , 1983 ; sasaki and epel , 1983 ; zimmerberg et al . , 1985 ) . stable conditions were achieved by waiting 10 min after shearing before initiating the solution exchange protocols . the origin of the decrease in calcium sensitivity during the first few minutes after shearing is not understood . intermediate calcium concentrations lead to stable , submaximal responses in the extent of fusion ( fig . 1 a ) . the loss of vesicles with time can not be described by a single exponential process . differences in the fusion time course after normalization of the fusion data to the submaximal extents of fusion indicate that the kinetics of the vesicles that take part in the fusion process depend upon the calcium concentration ( fig there appears to exist an absolute threshold in calcium concentration ( 5 m ) below which no vesicle fusion was observed . the fusion kinetics are faster , and higher calcium concentrations are required for comparable extents of fusion with l. pictus compared with s. purpuratus ( fig . 2 a ) . for each species studied , the transition from 0 to 100% fusion occurs over a narrow range of calcium concentrations . well after the time required to establish a spatially and temporally invariant calcium concentration , there exists an inflection point in the % fusion - vs .- time curve such that the instantaneous rate of fusion , d(%fusion)/dt , is maximum . the rate of fusion increases , peaks at this maximum rate , and then decreases in the presence of a defined , spatially and temporally invariant , concentration of calcium : the rate of fusion does not decrease monotonically with time ( fig . however , this kinetic feature can be obscured when the rate ( derivative ) curve is too noisy , the phase occurs within a time period comparable to the solution turnover in the chamber , or the temporal resolution is too low . for example , the early kinetic behavior of l. pictus was easily observed using a higher sampling rate ( 20 ms / point ) compared with the typical sampling rate ( 100 ms / point ) . the maximum rate of fusion in previously unchallenged cortices increases with increasing calcium concentration , while the time to reach the maximum rate decreases with increasing calcium concentration ( fig . the increase in the maximum rate of fusion with increasing calcium concentration is consistent with the previously observed increase in the inverse half time ( zimmerberg et al . , 1985 ) . what process(es ) could limit the extent and instantaneous rate of fusion in the continued presence of calcium ? the submaximal fusion response is not limited by an irreversible loss in the ability of the remaining vesicles to fuse . for example , the continued presence of elevated calcium for 30 min had no effect on subsequent fusion ( fig . 3 ) . in every preparation studied , the remaining population of vesicles fused after the addition of a high calcium concentration ( > 300 m ) , independent of the time and sequence of solution challenges . permanent inactivation of the remaining vesicles was never observed under the conditions described in this report . the calcium sensitivity of unfused vesicles surviving after submaximal stimulation is not altered by the continued presence of calcium . this rules out hypotheses relying on calcium - dependent irreversible transitions of the fusion apparatus to an inactive state . if the cessation of fusion was due to a reversible , calcium - dependent inactivation of the fusion machinery , then returning the preparation to a lower calcium concentration should reset the fusion process . upon reset , a second exposure to the initial calcium concentration should result in fusion of the remaining vesicles to the same fractional extent as observed previously . this property was not observed . once the stable , submaximal response was reached , enhanced fusion did not occur when cortices were challenged with a second application of the same calcium concentration after incubation in nominally zero calcium ( < 0.3 m ) from 0 to 30 min ( fig . the calcium sensitivity of unfused vesicles surviving after submaximal stimulation is not altered by the absence of calcium . the fusion process can not be reset and does not respond to the same calcium challenge elicited by two identical pulses separated in time by up to 30 min . the decrease in the instantaneous rate of fusion to zero in the continued presence of calcium , characteristic of a stable , submaximal extent of fusion , can not be attributed to reversible , calcium - dependent inactivation of the fusion apparatus . if the cessation of fusion occurs because a specific subpopulation of vesicles is removed through fusion ( heterogeneity ) , then only perfusion with higher calcium concentrations will enhance fusion . in the absence of other processes , the amount of fusion will be additive and only depend upon the final concentration of calcium reached . enhanced fusion was obtained , with or without an intervening chelator wash , only when the calcium concentration of the second challenge was raised . large variations in vesicle exocytosis among the 2040 cortices in the probed monolayer were not observed . the fusion rate observed after a second challenge of higher calcium concentration was variable relative to the fusion rate observed after a single challenge of the same final calcium concentration : rates after the second challenge were less than , equal to , or greater than those seen with single challenge experiments . nevertheless , the extent of fusion was additive , with the overall extent dependent only on the calcium concentration of the final challenge ( fig . 5 , and see fig . 7 a ) . in the isolated planar cortex , after a critical period during which removal of calcium is known to leave vesicles that are competent to fuse upon reapplication of the same calcium concentration ( zimmerberg et al . , 1985 ) , fusion to the stable , submaximal response is observed independent of the presence or absence of calcium . small ( < 4% ) differences in fusion were occasionally observed when the response to the first calcium challenge had not yet reached a plateau value at the time of the second calcium challenge ( fig . 6 a ) . the small changes in fusion that occur during this time could be mistakenly attributed to solution challenges in the absence of a continuous record of fusion . the absence of any abrupt change in fusion , with or without an intervening chelator wash , is consistent with the interpretation that the second challenge occurred during the time when previously activated vesicles , irreversibly committed to the fusion process , are fusing . in contrast , abrupt changes in fusion are observed only when calcium is removed before irreversible commitment to the fusion process ( fig . the importance of acquiring a continuous fusion record can not be overemphasized because changes in fusion observed during the calcium - independent period could be mistakenly attributed to additional interventions such as the removal or addition of a calcium challenge ( see discussion ) . the results from single and double challenges are summarized in the calcium activation curve ( fig . 7 a ) . the single and double challenge data were fit by a log - normal cumulative distribution function % fusion = 50 erfc{[pca + log ( 10 m)]/\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}(\sqrt{2}{\cdot}w)\}\end{equation*}\end{document } , where erfc is the complementary error function . for s. purpuratus , w = 0.23 0.03 and 0.24 0.06 , and midpoint m = 18.3 0.7 and 17.9 1.6 m ( n = 111 and 68 ) , respectively . for l. pictus , w = 0.25 0.03 and 0.25 0.05 , and midpoint m = 45.9 1.5 and 44.2 3.2 m ( n = 77 and 26 ) , respectively . for either species , the parameters of the fit for single and double challenge calcium activation curves were not significantly different ( p < 0.01 ) . with no evidence for different calcium activation curves arising from the sequence of calcium challenges , the entire data sets for s. purpuratus and l. pictus were fit using a log - normal cumulative distribution function with w = 0.23 0.02 and 0.24 0.02 , and midpoint m = 18.2 0.6 and 44.8 1.2 m , respectively . the shift in calcium sensitivity between the two species , m , is significant ( p < 0.05 ) , while the width of the underlying distribution , w , is invariant . the absence of any difference between the calcium activation curves derived from single and double challenges suggests that the exocytotic response was dependent only on the final calcium concentration , and not the sequence of concentration steps used to reach the final concentration . a log - normal probability density function was calculated from the log - normal cumulative distribution function ( fig . the first derivative of the calcium activation curve can be viewed as the underlying probability density function for fusion thresholds . a log - normal representation was chosen because calcium thresholds are exclusively positive values . the calcium concentration required for the first fusion event can be estimated from the probability density function for fusion thresholds . multiplying the probability density function by the total number of vesicles in a population approximates the frequency distribution of vesicle thresholds . an estimate of the calcium concentration required for the first fusion event occurs at a threshold frequency of one . for example , in a system of 20,000 vesicles , the first vesicle is estimated to fuse when the concentration of calcium exceeds 1 m in s. purpuratus and 7 m in l. pictus . this value is greater than the in vivo concentration of calcium in unfertilized eggs , regardless of species . the distribution of vesicle thresholds offers a rationale for explaining why vesicles fail to fuse in unfertilized eggs , where the calcium concentration is believed to be submicromolar ( reviewed in shen , 1995 ) : the lowest threshold for fusion is higher than the calcium concentration present . we conclude that the calcium sensitivity of cortical degranulation is heterogeneous : vesicle exocytosis ceases at a certain calcium concentration because all the vesicles that can fuse at that concentration have fused . irreversible and reversible inactivation of the fusion process was ruled out within our experimental times . this result is different from findings in permeabilized bovine adrenal medullary cells , where subsequent catecholamine release was obtained in a double challenge protocol with the same calcium concentration in both challenges ( baker and knight , 1981 ; knight and baker , 1982 ) . while the calcium - dependent processes of chromaffin vesicle exocytosis may differ from those of cortical vesicle exocytosis , it is likely , based on their time course for catecholamine release , that calcium was withdrawn from , and then returned to , the permeabilized chromaffin cells before the stable , submaximal response had reached its plateau ( e.g. , fig . 6 a , this plateau may have been difficult to identify in the absence of a continuous record of secretion , the presence of an endogenous ( nonstimulated ) secretion , long equilibration times , and interconversion between vesicle compartments . the failure to identify a single limiting process in the decay of the instantaneous rate other than a decrease in the number of vesicles in a selected population , the size of which is set by the calcium concentration , is consistent with a system having the property of earlier heterogeneity in calcium thresholds . , it is possible to invoke schemes having complicated rate , time , and concentration properties that can account for the behaviors that are observed . however , the existence of multiple subpopulations of vesicles with differing calcium thresholds is the simplest explanation for the behaviors observed in this system and represents our working hypothesis . that vesicles from the same cell type respond to calcium differently supports the hypothesis that vesicles that appear morphologically homogeneous can have different calcium thresholds ( ninomiya et al . , 1997 ) . we have proposed that the number and distribution of activated fusion complexes underlie the control of the rate and extent of triggered exocytosis , and that cortical vesicles have nine active fusion complexes , on average , at high calcium concentrations ( vogel et al . , 1996 ) . since fusion of a single vesicle is an all - or - none event that can be caused by one activated fusion complex , the probability density function for calcium thresholds can be regarded as the widest possible distribution of the lowest threshold values present on cortical vesicles . since a vesicle may have multiple activated fusion complexes , the lowest threshold group represents a subpopulation of the total number of activable fusion complexes present in the system . the sampling of the transition region of the calcium activity curve is not sufficient to distinguish between a continuous or discrete probability density function for calcium thresholds . however , there are at least five , experimentally defined , subpopulations with different calcium thresholds . that cortical vesicles are heterogeneous in their response to calcium raises questions on the molecular origin of this property . fusion may proceed through chemically identical complexes whose calcium sensitivity is dependent , before the introduction of calcium , on the local environment . for example , after insertion into various membranes and lipid preparations , the affinity of purified muscarinic acetylcholine receptors for antagonists is modulated by the lipid environment ( berstein et al . , 1989 ) . the degree of association between the vesicle and target membrane , local ionic conditions ( mclaughlin and whitaker , 1988 ) , and local lipid composition ( vogel et al . , 1993 ; reviewed in chernomordik et al . , 1995 ) may influence the fusion process and manifest as differing sensitivities to calcium . the heterogeneity in calcium sensitivity may arise from different proteins ( e.g. , a multigene family as in olfaction ; reviewed in chess et al . , 1992 ) , different protein isoforms ( e.g. , multiple synaptotagmins ; reviewed in littleton and bellen , 1995 ) , or different posttranslational modifications such as phosphorylation of calcium binding sites . efforts have been made to estimate the number of calcium ions involved in the fusion process based on models that relate the calcium concentration and fusion response . a fundamental assumption in these analyses is that fractional response is directly proportional to fractional calcium binding . the nonlinear relationship between calcium concentration and neurotransmitter release at the neuromuscular junction has been attributed to the cooperative action of four calcium ions in the release process ( dodge and rahamimoff , 1967 ) . dodge and rahamimoff ( 1967 ) recognized that their data were compatible with either a stochastic ( random ) model or with a stoichiometric reaction involving four calcium ions and four independent binding sites ( a single site binding curve raised to the fourth power ) . the nonlinear relationship in the squid giant axon ( augustine and charlton , 1986 ) was described by either a fourth order stoichiometric relationship or a hill equation with cooperative interactions between four binding sites . both dodge and rahamimoff ( 1967 ) and augustine and charlton ( 1986 ) relied on the linearity of n root double - reciprocal plots to conclude that power functions of a single site binding curve described the data . in these studies , the number of calcium ions is represented by a parameter that describes the steepness of the transition from 0 to 100% activity . however , we have now demonstrated that the response to calcium is consistent with multiple subpopulations with differing thresholds . these subpopulations exhibit receptor reserve behavior ( vogel et al . , 1996 ) . this implies that the underlying subpopulations , each with a different threshold , must have even steeper individual transitions than the overall measured response because the width of the measured response includes contributions from both the distribution of thresholds and the intrinsic width of each subpopulation . for the first time , we can identify multiple stable subpopulations whose existence requires that their individual transitions be steeper than the measured response . a steeper individual transition is consistent with a fusion process that requires the action of an even greater number of calcium ions than the number suggested by homogeneous models . the calculated dependence on calcium in other calcium - triggered exocytotic systems may be in error if a distribution of thresholds ( subpopulations ) exist in these systems . the involvement of many calcium ions may be a consequence of both protein and lipid binding processes . in fact , six individual transitions , each requiring the interaction of 10 calcium ions , results in an overall calcium activation curve that , when analyzed using homogeneous models , would suggest the interaction of three to four calcium ions . the assumption that fractional response is directly proportional to fractional calcium binding must be tested . although shifted , the invariance in the shape of the calcium activation curves for s. purpuratus and l. pictus implies that the cortical vesicle subpopulations in both species respond to the same increment in calcium , pca . similarities in the calcium activation curves for secretion from endocrine , exocrine , neuronal , and paracrine cells ( knight and scrutton , 1986 ) support the hypothesis that subpopulations of exocytotic vesicles exist in every calcium - triggered exocytotic system and that all subpopulations respond to approximately the same increment in calcium , pca . that equivalent subpopulations of vesicles enter the fusion process for comparable changes in calcium , pca , suggests that the molecular machinery involved in the overall fusion process performs the same work in overcoming a common fusion energy barrier , efusion , but that the initial and final energy levels differ between systems . these energy differences may be reflected in the calcium - binding properties of the calcium sensor and/or fusogenic apparatus and the lipid organization of the membranes . the two species , s. purpuratus and l. pictus , are found in the same environment but undergo the cortical reaction during the winter and summer , respectively . could the shift in the calcium activity curve towards higher calcium concentrations for l. pictus be a compensatory mechanism that prevents thermally driven fusion events , which may arise as a result of increased ambient temperature , from occurring ? in summary , we have determined that the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous . the cessation of fusion in the continued presence of calcium was not due to calcium - dependent inactivation . the dependence of the extent of exocytosis on calcium suggests that many calcium - dependent events are required for the fusion process . thus , calcium activation curves may arise from both an underlying distribution of calcium thresholds and multiple calcium - dependent events . irreversible and reversible inactivation of the fusion process was ruled out within our experimental times . this result is different from findings in permeabilized bovine adrenal medullary cells , where subsequent catecholamine release was obtained in a double challenge protocol with the same calcium concentration in both challenges ( baker and knight , 1981 ; knight and baker , 1982 ) . while the calcium - dependent processes of chromaffin vesicle exocytosis may differ from those of cortical vesicle exocytosis , it is likely , based on their time course for catecholamine release , that calcium was withdrawn from , and then returned to , the permeabilized chromaffin cells before the stable , submaximal response had reached its plateau ( e.g. , fig . 6 a , this work ; fig . 12 a , baker and knight , 1981 ) . this plateau may have been difficult to identify in the absence of a continuous record of secretion , the presence of an endogenous ( nonstimulated ) secretion , long equilibration times , and interconversion between vesicle compartments . the failure to identify a single limiting process in the decay of the instantaneous rate other than a decrease in the number of vesicles in a selected population , the size of which is set by the calcium concentration , is consistent with a system having the property of earlier heterogeneity in calcium thresholds . , it is possible to invoke schemes having complicated rate , time , and concentration properties that can account for the behaviors that are observed . however , the existence of multiple subpopulations of vesicles with differing calcium thresholds is the simplest explanation for the behaviors observed in this system and represents our working hypothesis . that vesicles from the same cell type respond to calcium differently supports the hypothesis that vesicles that appear morphologically homogeneous can have different calcium thresholds ( ninomiya et al . , 1997 ) . we have proposed that the number and distribution of activated fusion complexes underlie the control of the rate and extent of triggered exocytosis , and that cortical vesicles have nine active fusion complexes , on average , at high calcium concentrations ( vogel et al . , 1996 ) . since fusion of a single vesicle is an all - or - none event that can be caused by one activated fusion complex , the probability density function for calcium thresholds can be regarded as the widest possible distribution of the lowest threshold values present on cortical vesicles . since a vesicle may have multiple activated fusion complexes , the lowest threshold group represents a subpopulation of the total number of activable fusion complexes present in the system . the sampling of the transition region of the calcium activity curve is not sufficient to distinguish between a continuous or discrete probability density function for calcium thresholds . however , there are at least five , experimentally defined , subpopulations with different calcium thresholds . that cortical vesicles are heterogeneous in their response to calcium raises questions on the molecular origin of this property . fusion may proceed through chemically identical complexes whose calcium sensitivity is dependent , before the introduction of calcium , on the local environment . for example , after insertion into various membranes and lipid preparations , the affinity of purified muscarinic acetylcholine receptors for antagonists is modulated by the lipid environment ( berstein et al . , 1989 ) . the degree of association between the vesicle and target membrane , local ionic conditions ( mclaughlin and whitaker , 1988 ) , and local lipid composition ( vogel et al . , 1993 ; reviewed in chernomordik et al . , 1995 ) may influence the fusion process and manifest as differing sensitivities to calcium . the heterogeneity in calcium sensitivity may arise from different proteins ( e.g. , a multigene family as in olfaction ; reviewed in chess et al . , 1992 ) , different protein isoforms ( e.g. , multiple synaptotagmins ; reviewed in littleton and bellen , 1995 ) , or different posttranslational modifications such as phosphorylation of calcium binding sites . efforts have been made to estimate the number of calcium ions involved in the fusion process based on models that relate the calcium concentration and fusion response . a fundamental assumption in these analyses is that fractional response is directly proportional to fractional calcium binding . the nonlinear relationship between calcium concentration and neurotransmitter release at the neuromuscular junction has been attributed to the cooperative action of four calcium ions in the release process ( dodge and rahamimoff , 1967 ) . dodge and rahamimoff ( 1967 ) recognized that their data were compatible with either a stochastic ( random ) model or with a stoichiometric reaction involving four calcium ions and four independent binding sites ( a single site binding curve raised to the fourth power ) . the nonlinear relationship in the squid giant axon ( augustine and charlton , 1986 ) was described by either a fourth order stoichiometric relationship or a hill equation with cooperative interactions between four binding sites . both dodge and rahamimoff ( 1967 ) and augustine and charlton ( 1986 ) relied on the linearity of n root double - reciprocal plots to conclude that power functions of a single site binding curve described the data . in these studies , the number of calcium ions is represented by a parameter that describes the steepness of the transition from 0 to 100% activity . however , we have now demonstrated that the response to calcium is consistent with multiple subpopulations with differing thresholds . these subpopulations exhibit receptor reserve behavior ( vogel et al . , 1996 ) . this implies that the underlying subpopulations , each with a different threshold , must have even steeper individual transitions than the overall measured response because the width of the measured response includes contributions from both the distribution of thresholds and the intrinsic width of each subpopulation . for the first time , we can identify multiple stable subpopulations whose existence requires that their individual transitions be steeper than the measured response . a steeper individual transition is consistent with a fusion process that requires the action of an even greater number of calcium ions than the number suggested by homogeneous models . the calculated dependence on calcium in other calcium - triggered exocytotic systems may be in error if a distribution of thresholds ( subpopulations ) exist in these systems . the involvement of many calcium ions may be a consequence of both protein and lipid binding processes . in fact , six individual transitions , each requiring the interaction of 10 calcium ions , results in an overall calcium activation curve that , when analyzed using homogeneous models , would suggest the interaction of three to four calcium ions . the assumption that fractional response is directly proportional to fractional calcium binding must be tested . although shifted , the invariance in the shape of the calcium activation curves for s. purpuratus and l. pictus implies that the cortical vesicle subpopulations in both species respond to the same increment in calcium , pca . similarities in the calcium activation curves for secretion from endocrine , exocrine , neuronal , and paracrine cells ( knight and scrutton , 1986 ) support the hypothesis that subpopulations of exocytotic vesicles exist in every calcium - triggered exocytotic system and that all subpopulations respond to approximately the same increment in calcium , pca . that equivalent subpopulations of vesicles enter the fusion process for comparable changes in calcium , pca , suggests that the molecular machinery involved in the overall fusion process performs the same work in overcoming a common fusion energy barrier , efusion , but that the initial and final energy levels differ between systems . these energy differences may be reflected in the calcium - binding properties of the calcium sensor and/or fusogenic apparatus and the lipid organization of the membranes . the two species , s. purpuratus and l. pictus , are found in the same environment but undergo the cortical reaction during the winter and summer , respectively . could the shift in the calcium activity curve towards higher calcium concentrations for l. pictus be a compensatory mechanism that prevents thermally driven fusion events , which may arise as a result of increased ambient temperature , from occurring ? in summary , we have determined that the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous . the cessation of fusion in the continued presence of calcium was not due to calcium - dependent inactivation . the dependence of the extent of exocytosis on calcium suggests that many calcium - dependent events are required for the fusion process . thus , calcium activation curves may arise from both an underlying distribution of calcium thresholds and multiple calcium - dependent events . four different calcium concentrations are represented ( 35 , 14 , 10 , and 5 m ) . in this example , a calcium concentration of 5 m was below the threshold for fusion . the data were collected using a sample time of 0.1 s. ( b ) normalization of the fusion data to the submaximal extents of fusion for 10 , 14 , and 35 m calcium . differences in the fusion time course after normalization indicate that the kinetics of the vesicles that take part in the fusion process depend upon the calcium concentration . ( a ) percent fusion vs. time , species differences . for comparable extents of fusion , the data were collected using a sample time of 0.02 ( l. pictus ) and 0.1 ( s. purpuratus ) s. ( b ) rate , d(%fusion)/dt vs. time in the continued presence of calcium . the maximum rate of fusion is larger and occurs earlier in l. pictus compared with s. purpuratus . the data were low - pass filtered with a 1.0 hz cutoff frequency before calculating the derivative using third order forward differences . submaximal responses were maintained during the exposure of cortices to solutions containing 5 or 16 m . the smaller arrow represents the addition of either 5 or 16 m calcium while the larger arrow represents the calcium concentration ( > 300 m ) used to fuse all the remaining vesicles . the presence of elevated calcium for 30 min had no effect on subsequent fusion resulting from exposure to a higher concentration of calcium . enhanced fusion does not occur when cortices are challenged with a second application of 24 m calcium , s. purpuratus . cortices were washed and incubated in zero calcium for 0.5 , 1.0 , 3.0 , 10.0 , and 30 min ( a e ) before the second calcium challenge . the upward and downward arrows signify addition and removal of calcium , respectively . the largest arrow represents the calcium concentration ( > 300 m ) used to fuse all remaining vesicles . the sample time was 0.15 s. additivity in the extent of fusion , s. purpuratus . the final extent of fusion is independent of the sequence of concentrations used to elicit the response . the calcium concentrations were 14 , 24 , and > 300 m ( dp ) ; 24 and > 300 m ( sp ) . for clarity , the sample time was 0.15 s. ( a ) calcium activation curves for s. purpuratus and l. pictus , average values ( sem ) . unchallenged cortices are represented by solid symbols and previously challenged cortices are represented by open symbols . the solid lines are the log - normal cumulative distribution function , % fusion = 50 erfc{[pca + log ( 10 m)]/\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}(\sqrt{2}{\cdot}w)\}\end{equation*}\end{document } , with w = 0.23 0.02 and 0.24 0.02 and midpoint m = 18.2 0.6 and 44.8 1.2 m for s. purpuratus and l. pictus , respectively . ( b ) distribution of calcium thresholds ( probability density function ) for s. purpuratus and l. pictus derived from the derivative of the log - normal cumulative distribution function . the total area under each curve is one , corresponding to 100% fusion . ( a ) fusion can occur during solution exchange , before establishing the stable , submaximal response , which is independent of the presence or absence of calcium . the upward and downward arrows signify addition and removal of 24 m calcium , s. purpuratus . the absence of abrupt changes in fusion ( inset ) indicates that the solution exchange occurred during a time when previously activated vesicles are now fusing . t1 and the sample time was 0.15 s. ( b ) abrupt changes in fusion are observed only when calcium is removed before irreversible commitment to the fusion process . the upward and downward arrows signify addition and removal ( interrupted ) of 70 m calcium , s. purpuratus . the sample times were 0.15 and 0.05 s for uninterrupted and interrupted , respectively .
a graded response to calcium is the defining feature of calcium - regulated exocytosis . that is , there exist calcium concentrations that elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse . the role of calcium - dependent inactivation in defining the calcium sensitivity of sea urchin egg secretory vesicle exocytosis in vitro was examined . the cessation of fusion in the continued presence of calcium was not due to calcium - dependent inactivation . rather , the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous . any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent upon the magnitude of the calcium stimulus . the existence of multiple , stable subpopulations of vesicles is consistent with a fusion process that requires the action of an even greater number of calcium ions than the numbers suggested by models based on the assumption of a homogeneous vesicle population .
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Proceed to summarize the following text: the number of cardiac rhythm device implantations , including implantable cardioverter defibrillator ( icd ) and cardiac resynchronisation therapy ( crt ) devices , has increased fast in the past decade due to expanding indications and ageing of the population . although the benefits of these devices were demonstrated in randomised controlled trials , this concerned selected patients and real - life data are necessary to assess cardiac device implantation - related risks . several prospective and retrospective studies reported both short- and long - term complications related to device implantation and pacing system upgrade . however , the majority of these reports are derived from randomised clinical trials which reflect selected patients and circumstances whereas other studies concern relatively old reports , also including leads with passive fixation , which are nowadays less commonly used [ 28 ] . the objective of this prospective registry study was to assess the real - life incidence of lead dislodgement , malfunctioning or perforation during the first year following implantation in a dutch high - volume teaching hospital . data on all patients who underwent procedures of de novo cardiac rhythm device implantations and pacing system upgrades in our hospital between january 2010 and december 2011 were prospectively collected . the indications for the implantation of pacemakers , icd and crt devices were based on contemporary guidelines [ 9 , 10 ] . the procedures were performed by seven operators including two cardiac electrophysiology fellows under direct supervision of an attending cardiac electrophysiologist , in cardiac catheterisation laboratories equipped according to the guidelines of the european society of cardiology ( 9 ) . the baseline characteristics of patients including age , gender , left ventricular ejection fraction ( lvef ) , presence of conduction disorders and functional new york heart association ( nyha ) class were recorded in the prospective database . to identify the lead - related complications the database was searched for re - intervention procedures during the first year following the implantation . in all lead - related re - intervention cases , data on the clinical manifestation and course , results on chest x - ray , echocardiography and technical data on lead performance were collected . lead dislodgement was defined if there was documentation of a change in the lead tip position on chest x - ray and changes in electrical lead parameters ( rise in impedance , loss of sensing and pacing ) . lead electrical malfunctioning was defined if lead impedance , electrogram amplitude or threshold had changed abruptly necessitating surgical revision without clear changes in the position of the lead on chest x - ray . lead perforation was defined in case of high suspicion of cardiac perforation , e.g. an acute stabbing chest pain or dyspnoea , significant changes in electrical lead parameters and a significant amount of pericardial effusion requiring pericardiocentesis with or without extracardiac lead location on x - ray . screw perforation was defined if there were pericarditis - like symptoms without clear changes in electrical lead parameters and absence of a significant amount of pericardial effusion . continuous variables are expressed as mean sd and p value was calculated by using the anova test . categorical variables were presented as numbers and percentages and significance of differences were analysed using the test or fisher s exact test . the denoted p values were two - sided and p < 0.05 was considered significant . continuous variables are expressed as mean sd and p value was calculated by using the anova test . categorical variables were presented as numbers and percentages and significance of differences were analysed using the test or fisher s exact test . the denoted p values were two - sided and p < 0.05 was considered significant . during a period of 2 years , 1929 cardiac rhythm devices and 3909 leads were implanted in 1929 consecutive patients with commonly accepted indications for either pacemaker , icd or crt device implantation . the baseline characteristics of patients and type of device implantation are summarised in table 1 . patients with pacemaker indications were older than icd patients and 66 % of patients with cardiac rhythm device implantations were male . jude medical , biotronik , sorin group and boston scientific . of all implanted devices , 1148 ( 60 % ) were icds and 1148 ( 29 % ) of all implanted leads were icd leads . details regarding implanted devices and leads are summarised in figs . 1 and 2.table 1clinical characteristicsbaselinesingle - chamber pm n = 259dual - chamber pm n = 426single chamber icd n = 287dual chamber icd n = 364crt - p n = 91crt - d n = 504 p - valueage ( year)78 1174 1161 1365 1172 1269 9 p < 0.001male ( % ) 555572.5776576 p < 0.001lvef ( % ) 30 931 1240 1326 11 p < 0.001nyha class2.1 0.52.2 0.62.3 0.92.4 1 p = 0.1qrs duration ( ms)112 33112 40143 60161 59 p < 0.001p value is calculated by using anova test crt cardiac resynchronisation therapy , icd implantable cardioverter defibrillator , lvef left ventricular ejection fraction , nyha class new york heart association functional class , pm pacemakerfig . 2a total of 3909 leads implanted clinical characteristics p value is calculated by using anova test crt cardiac resynchronisation therapy , icd implantable cardioverter defibrillator , lvef left ventricular ejection fraction , nyha class new york heart association functional class , pm pacemaker a total of 1929 cardiac rhythm devices implanted a total of 3909 leads implanted thirty - one ( 1.5 % ) patients were re - admitted because of cardiac device infection , and all had explantation of their device . these consisted predominantly of micro - organisms that are part of the skin flora ( 84 % ) . a total of 90 ( 2.3 % of the leads ) lead - related complications occurred in 86 ( 4.4 % ) patients for which re - intervention was needed . the cause of re - intervention was mainly lead dislodgement ( 66 % ) , followed by malfunctioning ( 20 % ) and perforation ( 18 % ) . re - intervention was more common in men compared with women ( 70 % vs 30 % p = 0.001 ) . younger patients more often had re - intervention ( mean age 65 13 vs 70 13 years , p = 0.001 ) . a total of 3909 leads were implanted . during the first year of follow - up a total of 74 ( 1.9 % ) lead dislodgements or malfunctions occurred in 71 ( 3.7 % ) patients : 57 ( 1.4 % ) dislodgements and 17 ( 0.5 % ) malfunctions . regarding lead dislodgement , the right atrial ( ra ) lead ( 1.9 % ) showed the most frequent lead dislodgement compared with the right ventricular ( rv ) pacemaker lead ( 0.3 % ) or icd lead ( 1.8 % ) ( p = 0.0007 and p = 0.002 ) , table 2 . only 6 ( 1 % ) coronary sinus ( cs ) leads dislocated requiring re - intervention . regarding lead malfunction , the icd lead ( 0.8 % ) showed the most frequent lead malfunction compared with the ra lead ( 0.1 % ) ( p = 0.002 ) , table 2 . the timing of occurrence of lead dislodgement / malfunction is summarised in fig . 3 . the majority of ra and rv lead dislodgements occurred before discharge whereas the majority of ra and rv lead malfunction occurred after the 2nd month following implantation . all cs lead dislodgements or malfunctions occurred after the 2nd month following implantation . in almost 1/3 of the cases the sleeves on the leads were not fixed adequately and in 2/3 of the cases the cause of dislodgement was unclear . all re - do interventions were conducted without further complications.table 2lead dislodgement , malfunctioning and perforationtype of leadnumber implanteddislodgement ( % ) malfunctioning ( % ) lead perforation ( % ) with pericardiocentesisscrew perforation ( % ) without pericardiocentesisatrial leads144228 ( 1.9 ) 2 ( 0.1)5 ( 0.3)2 ( 0.1)rv pacemaker leads7242 ( 0.3)4 ( 0.5)1 ( 0.1)7 ( 0.9)icd leads114821 ( 1.8 ) 9 ( 0.8)1 ( 0.08)coronary sinus leads5956 ( 1 ) 2 ( 0.3)total390957 ( 1.5)17 ( 0.4)6 ( 0.15)10 ( 0.25)p value is calculated by using fisher exact test icd implantable cardioverter defibrillator , rv right ventricular p = 0.0007 compared with rv pacemaker lead as reference p = 0.002 compared with rv pacemaker lead as reference p = 0.15 compared with rv pacemaker lead as referencefig . 3timing of either lead dislodgement , malfunctioning or perforation . the majority of lead dislodgements occurred before discharge and all perforations occurred after the 2nd month of implantation . ld 1 lead dislodgement before discharge , lm 1 lead malfunctioning before discharge , lp 1 lead perforation before discharge , ld 2 lead dislodgement between discharge and 2 months , lm 2 lead malfunctioning between discharge and 2 months , lp 2 lead perforation between discharge and 2 months , ld 3 lead dislodgement between 2 months and 1 year , lm 3 lead malfunctioning between 2 months and 1 year , lp 3 lead perforation between 2 months and 1 year lead dislodgement , malfunctioning and perforation p value is calculated by using fisher exact test icd implantable cardioverter defibrillator , rv right ventricular p = 0.0007 compared with rv pacemaker lead as reference p = 0.002 compared with rv pacemaker lead as reference p = 0.15 compared with rv pacemaker lead as reference timing of either lead dislodgement , malfunctioning or perforation . the majority of lead dislodgements occurred before discharge and all perforations occurred after the 2nd month of implantation . ld 1 lead dislodgement before discharge , lm 1 lead malfunctioning before discharge , lp 1 lead perforation before discharge , ld 2 lead dislodgement between discharge and 2 months , lm 2 lead malfunctioning between discharge and 2 months , lp 2 lead perforation between discharge and 2 months , ld 3 lead dislodgement between 2 months and 1 year , lm 3 lead malfunctioning between 2 months and 1 year , lp 3 lead perforation between 2 months and 1 year in this study 595 crt - d / p devices were implanted . in the crt - d group , significantly more lead dislodgments or malfunctions were observed compared with the single - chamber pacemaker ( 30 versus 3 , p = 0.006 ) . the risk of any lead dislodgement or malfunctioning was higher in crt - d ( 5 % , p = 0.006 ) and dual - chamber icd ( 5.8 % , p = 0.002 ) as compared with single - chamber pacemaker ( 1.2 % ) . numbers of lead dislodgements per type of device are summarised in table 3.table 3lead dislodgement and type of devicetype of devicenumber of lead dislodgement or malfunctionrisk of any leads lead dislodgement or malfunctionnumber of lead / screw perforation with or without pericardiocentesisrisk of lead / screw perforation with or without pericardiocentesissingle chamber pm31.2 % 3 0.8 % dual chamber pm102.3 % 12 3.0 % single chamber icd10 3.5 % 10.3 % dual chamber icd21 5.8 % 00 % crt - d / p30 5.0 % 00 % crt cardiac resynchronisation therapy , icd implantable cardioverter defibrillator , pm pacemaker p = 0.09 compared with single chamber pacemaker as reference p = 0.002 compared with single chamber pacemaker as reference p = 0.006 compared with single chamber pacemaker as reference p = 0.35 compared with single chamber icd as reference p = 0.02 compared with single chamber icd as reference lead dislodgement and type of device crt cardiac resynchronisation therapy , icd implantable cardioverter defibrillator , pm pacemaker p = 0.09 compared with single chamber pacemaker as reference p = 0.002 compared with single chamber pacemaker as reference p = 0.006 compared with single chamber pacemaker as reference p = 0.35 compared with single chamber icd as reference p = 0.02 compared with single chamber icd as reference during follow - up , 16 leads ( 0.4 % of the leads ) showed screw / lead perforation in 15 ( 0.8 % ) patients . they were re - admitted to the hospital with pericarditis - like symptoms suspected for cardiac perforation . the clinical presentation of all suspected cardiac perforations was subacute ( 21 days after implantation ) . fourteen patients were implanted with single- or dual - chamber pacemaker and one patient with single - chamber icd . the majority of these patients were re - admitted after the first week of implantation ( fig . ten patients were re - admitted with pericarditis - like symptoms with a slight amount ( < 0.5 cm ) of pericardial effusion on the echocardiogram . in these patients only repositioning of the lead was sufficient without need for pericardiocentesis . the other five patients were re - admitted with symptoms of cardiac tamponade and a significant amount of pericardial effusion ( > 2 cm , with > 25 % respiratory mitral flow variation ) on the echocardiogram , suspect for lead perforation without obvious extracardiac location of a lead on chest x - ray . in these 5 ( 0.26 % ) patients , with dual - chamber pacemakers , pericardiocentesis was necessary and performed . in all cases a repositioning or implantation of a new lead was performed without further complications and without the need for thoracic surgical intervention . in all patients , the pericarditis - like symptoms disappeared after repositioning of the suspected lead . from the 16 ( 0.4 % ) lead / screw perforations , 7 were ra leads , 8 were rv pacemaker leads and 1 was an active fixation icd lead . rv pacemaker leads caused significantly more lead / screw perforation compared with icd leads ( p = 0.02 ) , table 3 . all rv leads causing perforations were located in rv apex region and four out seven ra leads which caused perforation were located in the lateral wall of the right atrium . most lead / screw perforations ( 12 cases ) occurred in dual - chamber pacemakers compared with single - chamber pacemakers ( 2 cases ) or single - chamber icd ( 1 case ) ( p = 0.02 ) . there were no lead / screw perforations in dual - chamber icd or crt - d / p devices . the rate for lead / screw perforation in dual - chamber pacemakers was 3 % , whereas this did not occur in crt - d / p patients . numbers of leads / screw perforations per type of device are summarised in table 3 . a total of 90 ( 2.3 % of the leads ) lead - related complications occurred in 86 ( 4.4 % ) patients for which re - intervention was needed . the cause of re - intervention was mainly lead dislodgement ( 66 % ) , followed by malfunctioning ( 20 % ) and perforation ( 18 % ) . re - intervention was more common in men compared with women ( 70 % vs 30 % p = 0.001 ) . younger patients more often had re - intervention ( mean age 65 13 vs 70 13 years , p = 0.001 ) . a total of 3909 leads were implanted . during the first year of follow - up a total of 74 ( 1.9 % ) lead dislodgements or malfunctions occurred in 71 ( 3.7 % ) patients : 57 ( 1.4 % ) dislodgements and 17 ( 0.5 % ) malfunctions . regarding lead dislodgement , the right atrial ( ra ) lead ( 1.9 % ) showed the most frequent lead dislodgement compared with the right ventricular ( rv ) pacemaker lead ( 0.3 % ) or icd lead ( 1.8 % ) ( p = 0.0007 and p = 0.002 ) , table 2 . only 6 ( 1 % ) coronary sinus ( cs ) leads dislocated requiring re - intervention . regarding lead malfunction , the icd lead ( 0.8 % ) showed the most frequent lead malfunction compared with the ra lead ( 0.1 % ) ( p = 0.002 ) , table 2 . the timing of occurrence of lead dislodgement / malfunction is summarised in fig . 3 . the majority of ra and rv lead dislodgements occurred before discharge whereas the majority of ra and rv lead malfunction occurred after the 2nd month following implantation . all cs lead dislodgements or malfunctions occurred after the 2nd month following implantation . in almost 1/3 of the cases the sleeves on the leads were not fixed adequately and in 2/3 of the cases the cause of dislodgement was unclear . all re - do interventions were conducted without further complications.table 2lead dislodgement , malfunctioning and perforationtype of leadnumber implanteddislodgement ( % ) malfunctioning ( % ) lead perforation ( % ) with pericardiocentesisscrew perforation ( % ) without pericardiocentesisatrial leads144228 ( 1.9 ) 2 ( 0.1)5 ( 0.3)2 ( 0.1)rv pacemaker leads7242 ( 0.3)4 ( 0.5)1 ( 0.1)7 ( 0.9)icd leads114821 ( 1.8 ) 9 ( 0.8)1 ( 0.08)coronary sinus leads5956 ( 1 ) 2 ( 0.3)total390957 ( 1.5)17 ( 0.4)6 ( 0.15)10 ( 0.25)p value is calculated by using fisher exact test icd implantable cardioverter defibrillator , rv right ventricular p = 0.0007 compared with rv pacemaker lead as reference p = 0.002 compared with rv pacemaker lead as reference p = 0.15 compared with rv pacemaker lead as referencefig . 3timing of either lead dislodgement , malfunctioning or perforation . the majority of lead dislodgements occurred before discharge and all perforations occurred after the 2nd month of implantation . ld 1 lead dislodgement before discharge , lm 1 lead malfunctioning before discharge , lp 1 lead perforation before discharge , ld 2 lead dislodgement between discharge and 2 months , lm 2 lead malfunctioning between discharge and 2 months , lp 2 lead perforation between discharge and 2 months , ld 3 lead dislodgement between 2 months and 1 year , lm 3 lead malfunctioning between 2 months and 1 year , lp 3 lead perforation between 2 months and 1 year lead dislodgement , malfunctioning and perforation p value is calculated by using fisher exact test icd implantable cardioverter defibrillator , rv right ventricular p = 0.0007 compared with rv pacemaker lead as reference p = 0.002 compared with rv pacemaker lead as reference p = 0.15 compared with rv pacemaker lead as reference timing of either lead dislodgement , malfunctioning or perforation . the majority of lead dislodgements occurred before discharge and all perforations occurred after the 2nd month of implantation . ld 1 lead dislodgement before discharge , lm 1 lead malfunctioning before discharge , lp 1 lead perforation before discharge , ld 2 lead dislodgement between discharge and 2 months , lm 2 lead malfunctioning between discharge and 2 months , lp 2 lead perforation between discharge and 2 months , ld 3 lead dislodgement between 2 months and 1 year , lm 3 lead malfunctioning between 2 months and 1 year , lp 3 lead perforation between 2 months and 1 year in this study 595 crt - d / p devices were implanted . in the crt - d group , significantly more lead dislodgments or malfunctions were observed compared with the single - chamber pacemaker ( 30 versus 3 , p = 0.006 ) . the risk of any lead dislodgement or malfunctioning was higher in crt - d ( 5 % , p = 0.006 ) and dual - chamber icd ( 5.8 % , p = 0.002 ) as compared with single - chamber pacemaker ( 1.2 % ) . numbers of lead dislodgements per type of device are summarised in table 3.table 3lead dislodgement and type of devicetype of devicenumber of lead dislodgement or malfunctionrisk of any leads lead dislodgement or malfunctionnumber of lead / screw perforation with or without pericardiocentesisrisk of lead / screw perforation with or without pericardiocentesissingle chamber pm31.2 % 3 0.8 % dual chamber pm102.3 % 12 3.0 % single chamber icd10 3.5 % 10.3 % dual chamber icd21 5.8 % 00 % crt - d / p30 5.0 % 00 % crt cardiac resynchronisation therapy , icd implantable cardioverter defibrillator , pm pacemaker p = 0.09 compared with single chamber pacemaker as reference p = 0.002 compared with single chamber pacemaker as reference p = 0.006 compared with single chamber pacemaker as reference p = 0.35 compared with single chamber icd as reference p = 0.02 compared with single chamber icd as reference lead dislodgement and type of device crt cardiac resynchronisation therapy , icd implantable cardioverter defibrillator , pm pacemaker p = 0.09 compared with single chamber pacemaker as reference p = 0.002 compared with single chamber pacemaker as reference p = 0.006 compared with single chamber pacemaker as reference p = 0.35 compared with single chamber icd as reference p = 0.02 compared with single chamber icd as reference during follow - up , 16 leads ( 0.4 % of the leads ) showed screw / lead perforation in 15 ( 0.8 % ) patients . they were re - admitted to the hospital with pericarditis - like symptoms suspected for cardiac perforation . the clinical presentation of all suspected cardiac perforations was subacute ( 21 days after implantation ) . fourteen patients were implanted with single- or dual - chamber pacemaker and one patient with single - chamber icd . the majority of these patients were re - admitted after the first week of implantation ( fig . ten patients were re - admitted with pericarditis - like symptoms with a slight amount ( < 0.5 cm ) of pericardial effusion on the echocardiogram . in these patients only repositioning of the other five patients were re - admitted with symptoms of cardiac tamponade and a significant amount of pericardial effusion ( > 2 cm , with > 25 % respiratory mitral flow variation ) on the echocardiogram , suspect for lead perforation without obvious extracardiac location of a lead on chest x - ray . in these 5 ( 0.26 % ) patients , with dual - chamber pacemakers , pericardiocentesis was necessary and performed . in all cases a repositioning or implantation of a new lead was performed without further complications and without the need for thoracic surgical intervention . in all patients , the pericarditis - like symptoms disappeared after repositioning of the suspected lead . from the 16 ( 0.4 % ) lead / screw perforations , 7 were ra leads , 8 were rv pacemaker leads and 1 was an active fixation icd lead . rv pacemaker leads caused significantly more lead / screw perforation compared with icd leads ( p = 0.02 ) , table 3 . all rv leads causing perforations were located in rv apex region and four out seven ra leads which caused perforation were located in the lateral wall of the right atrium . most lead / screw perforations ( 12 cases ) occurred in dual - chamber pacemakers compared with single - chamber pacemakers ( 2 cases ) or single - chamber icd ( 1 case ) ( p = 0.02 ) . there were no lead / screw perforations in dual - chamber icd or crt - d / p devices . the rate for lead / screw perforation in dual - chamber pacemakers was 3 % , whereas this did not occur in crt - d / p patients . numbers of leads / screw perforations per type of device are summarised in table 3 . in this prospective device complication registry of 1929 patients , 4.4 % of patients suffered from a lead dislodgement , malfunctioning or perforation during the first year following the implantation . the overall rate of lead dislodgement , malfunctioning or perforation requiring re - intervention was low ( 2.3 % of 3909 leads ) in our study . ra and icd leads were the leads with the highest risk ( 1.9 % ) of dislodgement compared with rv pacemaker and coronary sinus leads . the rate of coronary sinus lead dislodgement or malfunction within the first year following the implantation was very low in this study ( 1.4 % ) . dual - chamber icd implantation was the procedure with the highest risk of lead dislodgement and ddd pacemaker implantation with the highest risk of lead perforation . the overall lead dislodgement rate in our hospital is low and comparable ( 1.53.3 % ) with published studies [ 2 , 6 , 1014 ] . the crt trials [ 2 , 1315 ] reported any lead dislodgement rate varying from 2.9 to 10.6 % . in our hospital the rate of any lead dislodgement in crt - d / p devices was 5 % . in crt devices the lead failure rate exceeds the rate of lead failure in one- or two - chamber devices . the low rate of any lead dislodgements of 5 % in our group could be explained by a low rate of coronary sinus lead dislodgements . the rate of coronary sinus lead dislodgement in the present study is low ( 1.4 % ) compared with previous reports ( 4.08.4 % ) [ 16 , 17 ] . these properties enable the operators to choose the lead which fits best in the side branches of the coronary sinus . also the use of inner catheters and selective shackling of the side branches makes it easier to advance the lead inside the vessel and to achieve a stable position . almost all coronary sinus leads were implanted by three very experienced operators in our hospital . furthermore , since coronary sinus leads of all different manufactures are available in our hospital during the implantation , and there is no preferred manufacturer assigned to a case , the most suitable lead can be chosen to be implanted according to the anatomy , enhancing the success rate . we tried to identify the causes of lead dislodgments . in almost 1/3 of the cases the sleeves on the leads were not fixed adequately and in 2/3 of the cases the cause of dislodgement was unclear . this means that in 1/3 of the cases a lead dislocation could be prevented by adequate fixation . in the present study all lead / screw perforations were subacute and the incidence was very low ( 0.8 % ) and comparable with ( 0.65.2 % ) the published reports [ 1 , 5 , 7 ] . ddd pacemakers caused the most perforations ( 12 cases ) and the majority of perforated leads were rv leads . 1 ) all perforated rv leads were located in the rv apex which is the thinnest part of the right ventricle . in our routine practice we try to avoid the apex if possible and position the rv leads in a septal region if sufficient sensing and pacing values are present . 2 ) the relatively small size of the lead , 67 french , which more easily perforates the apex compared with the thick high - voltage icd ( 89 french ) lead and , according to laplace s law ( p = f / a ; pressure is the amount of force acting on a unit area ) , it is easy to understand that when the surface area is smaller , then the pressure will be higher on that surface area . 3 ) also the experience of operators plays an important role . in our teaching hospital most pacemakers were implanted by less experienced operators , cardiologists in training , and icds and crts by very experienced operators . the majority of perforated ra leads were located in the lateral wall , which is not the preferred site , due to unstable position or insufficient electrical signals and therefore the operator was forced to choose for this site . this study demonstrates the real - world common daily practice by implanting a large number of devices in a teaching hospital . meticulous longitudinal follow - up was performed , with documentation of complications in all patients . we identified the re - intervention due to lead - related complication when the patients were returned to cardiac catheterisation laboratories within first year of implantation . patients with lead - related complications who did not return for re - intervention based on the opinion of their cardiologists could be missed in our registry . furthermore , the follow - up period in the present study is no longer than 1 year . however , most lead dislocations and perforations are expected to occur within 1 year [ 1 , 7 , 11 ] . this is also supported by our own data . to obtain reliable information about the rate of complications , we used predefined definitions of complications . other device - related complications , including infection , were beyond the scope of this study . our analysis also did not evaluate mortality and duration of hospitalisation in the study population . in this large observational study , lead related re - intervention was necessary in 4.4 % of patients , more common in younger and male patients , and due to either lead dislodgement ( 66 % ) , malfunctioning ( 20 % ) or perforation ( 18 % ) . with a total of 3909 leads implanted , the incidence of lead dislodgement , malfunction or perforation was low ( 2.3 % ) . lead perforation was more common with rv leads , especially when placed in the apex . in the patients with lead perforation , pericardiocentesis was only necessary in 1/3 . with 1929 devices implanted , more lead complications were observed in dual - chamber icd and crt - d / p .
backgroundthe number of cardiac rhythm device implantations has been growing fast due to expanding indications and ageing of the population . complications of implantation were rare in the trials . however , these involved small numbers and selected patients . prospective real - life data are necessary to assess cardiac device implantation procedure - related risks.objectiveto determine the incidence and predictors of lead - related re - intervention in a dutch high - volume teaching hospital.methodsdata from all patients who underwent cardiac rhythm device implantation between january 2010 and december 2011 were collected in a prospective registry . at least 1 year of follow - up regarding re - intervention was available for all patients . lead - related reasons for re - intervention were categorised into lead dislodgement , malfunctioning or perforation.resultsone thousand nine hundred twenty - nine devices including 3909 leads were implanted . in 595 patients ( 30.8 % ) a crt - d / p was implanted . lead - related re - intervention was necessary in 86 ( 4.4 % ) patients ; it was more common in younger and male patients , and due to either lead dislodgement ( 66 % ) , malfunctioning ( 20 % ) or perforation ( 18 % ) . coronary sinus lead dislodgement or malfunctioning was 1.4 % . right atrial dislodgement ( 1.9 % , p < 0.001 ) or icd lead dislodgement ( 1.8 % , p = 0.002 ) was more common than right ventricular dislodgement ( 0.3 % ) . the incidence of lead malfunctioning was higher ( 0.8 % ) in icd leads . an apical position of the right ventricular lead and lateral wall position of the right atrial lead were related to cardiac perforation.conclusionsthe incidence of lead - related re - intervention was comparable with the literature . the majority of re - interventions were due to lead dislodgements , particularly with right atrial and icd leads . re - intervention due to coronary sinus lead dislodgement was rare .
You are an expert at summarizing long articles. Proceed to summarize the following text: anaerobic conditions result in post - traumatic changes in glucose metabolism that can have long - term consequences . for example , in chronic tbi studies , 6 months after patients suffered diffuse axonal injury , fluorodeoxyglucose positron emission tomography ( pet ) analysis shows bilateral regional decreases of resting glucose metabolism in the frontal and temporal lobes , thalamus , and cerebellum , compared with healthy gender - matched controls . in patients with persistent neuropsychological deficits , early after tbi , elevated serum glucose levels have been considered deleterious based on the assumption that presentation of the ischemic brain with high levels of glucose would drive anaerobic glycolysis , resulting in the accumulation of toxic levels of lactic acid . at least one study found that persistent hyperglycemia , defined as average daily blood glucose levels > 149 mg / dl for the first week after injury , was associated with a five - fold increase in mortality in a cohort of 834 patients with severe tbi . others have found that maintenance of blood glucose below 145 mg / dl significantly reduces lactate production , and results in optimal lactate / glucose and oxygen / glucose indices ( as determined from arterial - jugular venous lactate , and glucose and oxygen content differences ) following severe tbi , at least as compared to higher blood glucose levels . the mechanisms whereby glucose may worsen the ischemic threshold in neurons after injury are not well understood . in neuron cultures exposed to ischemic conditions , post - ischemic superoxide production and cell death these studies suggest that glucose is a requisite electron donor for reperfusion - induced neuronal superoxide production . moreover , investigations of the mechanisms of neuronal injury that result from hypoglycemic coma have led some to conclude that the injury from hypoglycemia actually occurs with reperfusion , where high blood glucose concentrations initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal nadph oxidase . because of the depression of aerobic metabolism following tbi , and associated mitochondrial dysfunction , elevation of brain tissue o2 tension has been shown to significantly reduce extracellular lactate concentrations and the lactate / pyruvate ratio . there is both clinical and experimental evidence that hyperglycemia lowers the ischemic neuronal threshold in the presence of neurological injury , and that lactic acidosis increases astrocytic cell death [ 8 - 10 ] . however , large clinical trials of therapies designed to induce a metabolic alkalosis [ tromethamine ( tham ) ] or respiratory alkalosis ( hyperventilation ) , based on the concern about post - traumatic lactic acidosis , did not find outcomes to be improved with these therapies . in fact , there was some evidence that outcomes were actually worse when patients received prophylactic hyperventilation therapy . based on the concern that even slightly elevated blood glucose levels can cause or exacerbate secondary brain injury , investigators have conducted clinical trials to test the efficacy of tight glucose control , or maintenance of blood glucose levels of less than 110 - 120 mg / dl by using continuous insulin infusions . in a prospective study of 240 adult patients with severe tbi , patients were randomly assigned either to a tight glucose control group ( intensive insulin therapy ) with glucose levels maintained at 80 mg / dl throughout their intensive care unit ( icu ) stay , or to a conventional glucose control group where insulin was not given unless serum glucose levels exceeded 200 mg / dl . the groups were well matched according to several measures of severity of injury and age . six - month mortality rates were similar for the two groups , though significantly more patients in the intensive insulin therapy group had good outcomes ( that is , a glasgow outcome scale ( gos ) score of 4 or 5 ) than did the conventional therapy patients ( 29.1% versus 22.4% , p < 0.05 ) . in addition , intensive insulin therapy was associated with a significantly lower infection rate and shorter icu stay . however , the incidence of severe hypoglycemia in the tight glucose control group was not reported . most of the other clinical studies of patients with tbi , as well as stroke and subarachnoid hemorrhage , have not found benefit from tight glucose control , and there is increasing concern about the associated risk of hypoglycemic episodes and the further neuronal injury that can result from hypoglycemia . episodes of hypoglycemia have been found to be associated with increased extracellular levels of glutamate , glycerol , and the lactate / pyruvate ratio , all measures of cellular distress . in one prospective observational study of 30 icu patients ( tbi , stroke , subarachnoid hemorrhage ) , the safety of continuous insulin infusions for maintaining blood glucose levels between 81 and 110 mg / dl was studied . adherence to the protocol was 71% , but blood glucose levels were maintained in the desired range for only 42% of the total protocol implementation time . consequently , 60% of the patients in this study experienced at least one hypoglycemic event . a retrospective comparison of 57 neuro - icu patients who had tight glucose control with 64 patients admitted to the icu prior to initiation of the protocol also found a higher incidence of hypoglycemia , as well as a longer icu stay , in the group with tight glucose control . a prospective study of 97 patients with severe tbi assigned either to a group targeted to blood glucose levels of 80 - 120 mg / dl using a continuous insulin infusion , or to a group that received insulin only if the blood glucose levels exceeded 220 mg / dl , also found that episodes of hypoglycemia , defined as a blood glucose < 80 mg / dl , were significantly more common for the tight glucose control group . although the duration of icu stay also was shorter for that group , there was no difference in icu infection rates , or in gos or mortality at 6 months . several investigators used microdialysis to examine the metabolic effects of tight glucose control , with the probes placed either in normal appearing brain , or brain adjacent to contusions . changes in dialysate levels of glucose , lactate , and pyruvate have been shown to correlate well with glucose metabolism as defined with fluorodeoxyglucose - pet . in 20 patients with severe tbi , continuous insulin infusions were used to maintain blood glucose levels of 80 - 120 mg / dl . patients with dialysate glucose levels < 0.7 mmol / l ( 12.6 mg / dl ) and lactate / pyruvate ratios > 40 were considered in brain energy crisis , and this condition was associated with a seven - fold increase in the odds of mortality at hospital discharge . compared with patients in whom the blood glucose was not tightly controlled ( 121 - 180 mg / dl ) , tight glucose control was associated with a significant increase in the incidence of dialysate glucose levels < 0.7 mmol / l ( 12.6 mg / dl ) and lactate / pyruvate ratios > 40 ( brain energy crisis ) . in similar studies at the university of california , los angeles , 14 tbi patients acutely managed with intensive insulin therapy were compared with 33 tbi patients treated with a intensive insulin therapy resulted in a 70% reduction of dialysate glucose levels , a significant increase in the lactate / pyruvate ratio , which frequently exceeded 40 , and increased levels of glutamate , another marker of cellular distress following trauma . significantly , more of the intensive - insulin - therapy patients had critically low extracellular glucose levels compared with those who had the loose insulin protocol . moreover , the percentage of time that the dialysate lactate / pyruvate ratio was above 40 was significantly correlated with the extent of frontal lobe brain atrophy at 6 months after injury . this was observed even in patients who had had normal appearing frontal lobes on their initial ct ( computed tomography ) scans immediately after injury . during the acute care of patients with severe tbi , there is some evidence that the optimal blood glucose level may change over time . retrospectively evaluated the influence of predefined blood glucose targets ( 63 - 117 mg / dl versus 90 - 144 mg / dl ) on intracranial pressure , mortality , and length of stay in the icu in 228 patients with severe tbi . they found that maintaining the blood glucose at 90 - 144 mg / dl was beneficial during the first week after injury primarily because there were significantly fewer hypoglycemic episodes in that group compared to the group for whom 63 - 117 mg / dl was the target . during the second week the group maintained at the lower target range had a reduced intracranial pressure , and decreased rates of pneumonia , bacteremia , and urinary tract infections compared to the higher blood glucose group . while glucose appears to be the primary metabolic substrate in the healthy brain , lactate may be the preferred energy substrate following tbi . in a rodent study of cerebral oxygen consumption following fluid percussion injury , it was found that glucose sustained mitochondrial respiration at a low level fixed rate , but increasing its concentration did not result in up - regulation of oxygen consumption . however , lactate administration resulted in a dose - dependent increase in mitochondrial oxygen consumption that may well have enabled the mitochondria to meet the increased energy needs of the injured brain . there also is experimental evidence that astrocytes contain low levels of glycogen , and that during hypoglycemia the astrocyte glycogen breaks down to lactate that is then transferred to adjacent neurons or axons and is used aerobically as fuel . ketones also have been proposed as an alternative fuel following tbi . in a murine study of controlled cortical impact injury , feeding the rat a ketogenic diet after injury led to a more rapid normalization of the cerebral metabolic rate for glucose ( cmrglucose ) , and decreased contusion volume . based on the concern that even slightly elevated blood glucose levels can cause or exacerbate secondary brain injury , investigators have conducted clinical trials to test the efficacy of tight glucose control , or maintenance of blood glucose levels of less than 110 - 120 mg / dl by using continuous insulin infusions . in a prospective study of 240 adult patients with severe tbi , patients were randomly assigned either to a tight glucose control group ( intensive insulin therapy ) with glucose levels maintained at 80 mg / dl throughout their intensive care unit ( icu ) stay , or to a conventional glucose control group where insulin was not given unless serum glucose levels exceeded 200 mg / dl . the groups were well matched according to several measures of severity of injury and age . six - month mortality rates were similar for the two groups , though significantly more patients in the intensive insulin therapy group had good outcomes ( that is , a glasgow outcome scale ( gos ) score of 4 or 5 ) than did the conventional therapy patients ( 29.1% versus 22.4% , p < 0.05 ) . in addition , intensive insulin therapy was associated with a significantly lower infection rate and shorter icu stay . however , the incidence of severe hypoglycemia in the tight glucose control group was not reported . most of the other clinical studies of patients with tbi , as well as stroke and subarachnoid hemorrhage , have not found benefit from tight glucose control , and there is increasing concern about the associated risk of hypoglycemic episodes and the further neuronal injury that can result from hypoglycemia . episodes of hypoglycemia have been found to be associated with increased extracellular levels of glutamate , glycerol , and the lactate / pyruvate ratio , all measures of cellular distress . in one prospective observational study of 30 icu patients ( tbi , stroke , subarachnoid hemorrhage ) , the safety of continuous insulin infusions for maintaining blood glucose levels between 81 and 110 mg / dl was studied . adherence to the protocol was 71% , but blood glucose levels were maintained in the desired range for only 42% of the total protocol implementation time . consequently , 60% of the patients in this study experienced at least one hypoglycemic event . a retrospective comparison of 57 neuro - icu patients who had tight glucose control with 64 patients admitted to the icu prior to initiation of the protocol also found a higher incidence of hypoglycemia , as well as a longer icu stay , in the group with tight glucose control . a prospective study of 97 patients with severe tbi assigned either to a group targeted to blood glucose levels of 80 - 120 mg / dl using a continuous insulin infusion , or to a group that received insulin only if the blood glucose levels exceeded 220 mg / dl , also found that episodes of hypoglycemia , defined as a blood glucose < 80 mg / dl , were significantly more common for the tight glucose control group . although the duration of icu stay also was shorter for that group , there was no difference in icu infection rates , or in gos or mortality at 6 months . several investigators used microdialysis to examine the metabolic effects of tight glucose control , with the probes placed either in normal appearing brain , or brain adjacent to contusions . changes in dialysate levels of glucose , lactate , and pyruvate have been shown to correlate well with glucose metabolism as defined with fluorodeoxyglucose - pet . in 20 patients with severe tbi , continuous insulin infusions were used to maintain blood glucose levels of 80 - 120 mg / dl . patients with dialysate glucose levels < 0.7 mmol / l ( 12.6 mg / dl ) and lactate / pyruvate ratios > 40 were considered in brain energy crisis , and this condition was associated with a seven - fold increase in the odds of mortality at hospital discharge . compared with patients in whom the blood glucose was not tightly controlled ( 121 - 180 mg / dl ) , tight glucose control was associated with a significant increase in the incidence of dialysate glucose levels < 0.7 mmol / l ( 12.6 mg / dl ) and lactate / pyruvate ratios > 40 ( brain energy crisis ) . in similar studies at the university of california , los angeles , 14 tbi patients acutely managed with intensive insulin therapy were compared with 33 tbi patients treated with a intensive insulin therapy resulted in a 70% reduction of dialysate glucose levels , a significant increase in the lactate / pyruvate ratio , which frequently exceeded 40 , and increased levels of glutamate , another marker of cellular distress following trauma . significantly , more of the intensive - insulin - therapy patients had critically low extracellular glucose levels compared with those who had the loose insulin protocol . moreover , the percentage of time that the dialysate lactate / pyruvate ratio was above 40 was significantly correlated with the extent of frontal lobe brain atrophy at 6 months after injury . this was observed even in patients who had had normal appearing frontal lobes on their initial ct ( computed tomography ) scans immediately after injury . during the acute care of patients with severe tbi , there is some evidence that the optimal blood glucose level may change over time . retrospectively evaluated the influence of predefined blood glucose targets ( 63 - 117 mg / dl versus 90 - 144 mg / dl ) on intracranial pressure , mortality , and length of stay in the icu in 228 patients with severe tbi . they found that maintaining the blood glucose at 90 - 144 mg / dl was beneficial during the first week after injury primarily because there were significantly fewer hypoglycemic episodes in that group compared to the group for whom 63 - 117 mg / dl was the target . during the second week the group maintained at the lower target range had a reduced intracranial pressure , and decreased rates of pneumonia , bacteremia , and urinary tract infections compared to the higher blood glucose group . while glucose appears to be the primary metabolic substrate in the healthy brain , lactate may be the preferred energy substrate following tbi . in a rodent study of cerebral oxygen consumption following fluid percussion injury fixed rate , but increasing its concentration did not result in up - regulation of oxygen consumption . however , lactate administration resulted in a dose - dependent increase in mitochondrial oxygen consumption that may well have enabled the mitochondria to meet the increased energy needs of the injured brain . there also is experimental evidence that astrocytes contain low levels of glycogen , and that during hypoglycemia the astrocyte glycogen breaks down to lactate that is then transferred to adjacent neurons or axons and is used aerobically as fuel . ketones also have been proposed as an alternative fuel following tbi . in a murine study of controlled cortical impact injury , feeding the rat a ketogenic diet after injury led to a more rapid normalization of the cerebral metabolic rate for glucose ( cmrglucose ) , and decreased contusion volume . while there is experimental evidence that elevated blood glucose levels can cause or exacerbate secondary brain injury , a critical issue yet to be resolved is that of cause or effect . in one large cohort of severely injured trauma patients , those with early hyperglycemia did have a two - fold higher mortality rate and a 30% higher incidence of multiple organ failure compared to those who did not . correlated admission glucose levels to 6-month outcomes in nearly 5,000 patients from six phase iii trials entered into the impact ( international mission for prognosis and analysis of clinical trials in tbi ) database , and found a significant association with increasing levels of admission blood glucose and poor outcomes . but in both of these studies , the increased blood glucose levels may have been related to the increased stress response associated with more severe injuries ; that is , elevated blood glucose levels were a reflection , but not the cause , of very severe injury . so while it is clear that with tbi , as well as with stroke and a number of other neurologic diseases , hyperglycemia is common soon after the injury , it is not clear that aggressive reduction of the elevated blood glucose will improve neurologic outcomes . it is increasingly apparent , however , that tight glucose control is not only associated with increased hypoglycemic episodes , but also with direct extracellular ( microdialysis ) evidence of cellular distress and brain energy crisis . in addition , there is evidence that lactate , the principle metabolite of anaerobic glycolysis , may be a preferred fuel for neurons after tbi . there even is evidence for an increase in brain metabolic demand early after injury that may lead to a deficiency in cerebral extracellular glucose . in conclusion , the majority of currently available clinical and preclinical evidence does not support tight glucose control ( maintenance of blood glucose levels below 110 - 120 mg / dl ) during the acute care of patients with severe tbi .
tight glucose control during the acute care of patients with severe traumatic brain injury has recently been advocated based on experimental concerns about deleterious effects of presenting the injured brain with a high glucose load , however , there is little or no clinical evidence that hyperglycemia worsens neurologic injury . the majority of the clinical studies of tight glucose control find that it is associated with an increased risk of hypoglycemic episodes and cellular injury , when compared to conventional glucose control protocols .
You are an expert at summarizing long articles. Proceed to summarize the following text: this condition can cause subfertility or infertility , and psychological trauma in men ( 1 , 2 ) . to prevent ischemic necrosis , treatment should be done as soon as possible . if the testicle can be detorsed within 6 hr , over 90% of patients experience testicular salvage , while success decreases to < 10% after 24 hr ( 3 ) . patients or their parents hesitate to consult doctors for a genital condition because of lack of medical knowledge . distance from home to a medical center and the economic status of the patients ' family may affect the delaying treatment ( 4 ) . etiology of testicular torsion is known as ' bell clapper ' anatomical deformity ( 5 ) . a family history of testicular torsion has been described as one of the etiology ( 6 ) . nationwide studies in the united states and taiwan reported incidences of testicular torsion in males aged 1 - 25 yr as 4.5 cases per 100,000 men per year overall , and 3.5 cases per 100,000 men per year in men < 25 yr of age ( 8 , 9 ) . however , information on koreans is lacking . we performed a nationwide epidemiological study on the epidemiology of testicular torsion in korean men . the korean urologic association operated the uropds ( urologic partitioned data set ) patient registry from 2006 to 2011 . uropds data consists of age , diagnosis , kind of surgery , time of admission and time of discharge . to calculate the incidence rate of testicular torsion in the general population , the total population was cited from the korean statistical information service . a national census was made in 2010 ; and the estimated population was determined every year from 2006 to 2011 . the average incidence of 6 yr was calculated by dividing the total population of males for the 6 yr by the total number of patients . testicular salvage rate was calculated in patients who had undergone orchiopexy divided by the total number of patients . age group was divided as infancy ( < 1 yr ) , children ( 1 - 11 yr ) , adolescents ( 12 - 20 yr ) , and adults ( < 20 yr ) ( 10 ) . pearson chi - square test was used to evaluate the association between variables and testicular salvage . this study was approved by the institutional review board of the samsung medical center in seoul , korea ( irb number , smc 2014 - 05 - 023 ) . pearson chi - square test was used to evaluate the association between variables and testicular salvage . this study was approved by the institutional review board of the samsung medical center in seoul , korea ( irb number , smc 2014 - 05 - 023 ) . from january 2006 to december 2011 , 1,591 cases were included in uropds by a keyword search using ' testicular torsion ' . by year , the number of patients with testicular torsion from 2006 to 2011 was 225 , 254 , 272 , 280 , 346 , and 213 , respectively . the incidence of testicular torsion in male was 1.1 per 100,000 and 2.9 per 100,000 in males < 25 yr old . incidence rate was decreased after the middle age , the disease can occur at any age ( table 2 ) . total testicular salvage rate was 75.7% overall and 75.4% for those < 25 yr of age . testicular salvage rate in infancy was 65.9% , which was lower than any other age group , but not statistically significant . testicular torsion is a well known emergency in urology and can occur at any age . our study shows that the incidence was 2.9 cases per 100,000 person years of males < 25 yr of age and 1.1 cases per 100,000 person years at all ages . the incidence of testicular torsion in males 1 - 25 yr of age has been reported as 4.5 cases per 100,000 male subjects per year in the united states ( 8) . a us study indicated that the estimated yearly incidence of testicular torsion for males < 18 yr of age was 3.8 per 100,000 ( 4 ) . another nationwide study in taiwan reported 3.5 cases per 100,000 person years for males < 25 yr of age ( 9 ) . an incidence of 1.4 cases per 100,000 men was reported in brazil ( 11 ) . it may reflect the clinical distinction between extravaginal torsion in newborns and infants and children ( 12 ) . many nationwide studies have been on the basis of the statistcs of the country and are classified based on international classification of disease ( icd ) . with these methods , there is a possibility of miscoding if symptoms are similar with testicular torsion . for example , miscoding can occur when diagnosis changes after scrotal exploration . in the case of the uropds data , a doctor needed to enter the data directly after final diagnosis . most testicular torsion patients visit a hospital presenting with scrotal pain , swelling , and erythema ( 13 ) . as a general rule , the decision depends on testicular color when a physician detorses the testis in the surgical field . a continuous black color , which means irreversible necrosis , is the most indicative of the need for testicle removal . delayed diagnosis and treatment can increase the rate of testicular removal . in this study , cost et al . ( 14 ) reported a 68.1% salvage rate from patients aged 1 - 17 yr old and zhao et al . ( 15 ) studied testicular salvage rate by ages of 17 - 20 and 21 - 34 yr . the salvage rate differed with 70.3% , of testes salvaged in the younger group versus only 41% in the older group . in taiwan , huang et al . ( 9 ) reported a rate of 74.4% in patients < 25 yr of age . testicular salvage in torsion depends on timely presentation , diagnosis , and surgical intervention . in other time sensitive disease , such as appendicitis and myocardial infarction , efforts to improve outcomes have traditionally focused on public awareness to expedite presentation and provider education to hasten diagnosis ( 16 , 17 ) . notably , there is similar testis salvage rate between rural and urban area in korea . in the us , ( 4 ) reported that age , insurance type , and living area influence testicular salvage rate . this better salvage rate in korea is presumed to be due to the result of small size of territory , dense distribution of hospitals , and national health insurance of korea ( nhi ) . the nhi system initially offered plans with low premiums , low benefits , and low doctors ' fees to contribute to eliminating inequities based on socioeconomic status . this approach improved access to medical services , eliminated gaps between urban and rural areas and between different classes with respect to the use of services ( 18 ) . in the future , korean urologists will likely decline due to decreased urology residents in korea , so similar longitudinal study design will be interesting in the future . this variable is involved to evaluate influence of medical accessibility but it is hard to reflect each patient 's medical accessibility . in conclusion , the incidence of testicular torsion in males aged < 26 yr was 2.9 cases per 100,000 men per year . approximately 75.7% patients with testicular torsion finally received orchiopexy in korea . to minimize the possibility of orchiectomy for testicular torsion , it is important to improve public awareness to expedite presentation and provider education to hasten diagnosis and surgery .
testicular torsion is a surgical emergency in the field of urology . knowledge of the epidemiology and pathophysiology is significant to an urologist . however , the epidemiology of testicular torsion in korea has not been studied . we performed a nationwide epidemiological study to improve knowledge of the epidemiology of testicular torsion . from 2006 - 2011 , the korean urologic association began the patient registry service . the annual number of patients with testicular torsion from 2006 to 2011 were 225 , 250 , 271 , 277 , 345 , and 210 , respectively . the overall incidence of testicular torsion in males was 1.1 per 100,000 ; however , the incidence in men less than 25 yr old was 2.9 per 100,000 . adolescents showed the highest incidence . total testicular salvage rate was 75.7% in this survey . there was no geographic difference of testicular salvage rate . minimizing the possibility of orchiectomy for testicular torsion is important to improve public awareness to expedite presentation and provider education to improve diagnosis and surgery.graphical abstract
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Proceed to summarize the following text: exosomes are extracellular vesicles 30100 nm in diameter that are shed by most cells . they were first observed in 1987 in maturing reticulocytes , and recently they have gained attention as agents of intercellular communication and as potential prognostic tools . in previous studies , we interrogated the content of myeloid - derived suppressor cells ( mdsc ) and exosomes shed by mdsc . we focused on these cells because of their widespread presence in most cancer patients and their critical role in promoting tumor progression through their inhibition of innate and adaptive antitumor immunity . we have reported that exosomes shed by mdsc contain proinflammatory molecules that drive the accumulation and immune suppressive potency of mdsc and macrophages , respectively , and have identified the specific proteins responsible for these bioactivities . on the basis of these and other potential functions , there is global interest in the nature of the protein cargo carried by exosomes . exosomes are formed by a series of intracellular events initiated by the invagination of the plasma membrane to form endosomes . within endosomes , these luminal vesicles and their cargo are then incorporated into the lysosome for degradation , recycled to the plasma membrane , or exocytosed as exosomes . ubiquitination has been shown to signal both the internalization of surface proteins and the sorting of endosomal proteins into luminal vesicles . given the strategic role of ubiquitination in intracellular protein trafficking , the present study seeks to confirm the presence and identify ubiquitinated proteins in exosomes derived from mdsc . a previous study using western blot analyses indicated that exosomes and their parental cells contain distinct populations of ubiquitinated proteins ; however , the conjugated proteins were not identified . identification of the conjugated proteins allows assignment of their original locations in the parent cell , their original cell functions , and the range of protein sizes and pi values and may contribute to understanding the complexity of the endosomal pathway . in addition , enriching for ubiquitinated proteins allows detection of proteins that may be relatively low in abundance but play an important role in exosome structure and function and that contribute to mdsc function . in the present work , ubiquitinated proteins have been recognized in mdsc - derived exosomes by identifying peptides that carry glycinylglycine - modified lysine residues as remnants of the ubiquitin carboxyl terminus . additionally , we have used gene ontology annotations and the uniprot database to look for trends in the source , function , size , and pi values of the ubiquitinated species . mass spectrometry - based bottom - up proteomics has proven to be a powerful tool for recognizing ubiquitinated tryptic peptides and identifying sites of ubiquitination on these peptides and their related proteins . tryptic digestion of ubiquitinated proteins cleaves ubiquitin at r74 , which leaves two glycine residues on the modified lysine of the substrate peptide . enrichment of ubiquitinated proteins from mdsc - derived exosomes was accomplished using two immunoprecipitation steps : immunoprecipitation of ubiquitinated proteins followed by tryptic digestion and immunoprecipitation of peptides containing glycinylglycine - modified lysine residues . in a parallel workflow , immunoprecipitation of ubiquitinated proteins was followed by 1d gel electrophoresis and in - gel digestion . in each case , ms / ms and a bioinformatic search program that allowed for the variable modification of glycinylglycine - modified lysine residues ( kgg ) . here these ubiquitinated proteins constitute a small subset of proteins in these exosomes , originate from a diversity of subcellular locations , and have a variety of functions . balb / c mice were injected in the mammary fat pad with approximately 7000 wild - type 4t1 mammary carcinoma cells stably transfected to express interleukin-1 ( il-1 ) . when tumors were greater than approximately 8 mm in diameter ( about 34 weeks after initial inoculation ) , mdsc were harvested from the blood , stained with fluorescently labeled monoclonal antibodies against markers of mdsc ( gr1 and cd11b ) , and analyzed by flow cytometry . cell populations that were greater than 90% gr1cd11b were used in all experiments . for each experiment , a total of about 1 10 mdsc were pooled from 2 to 3 mice . the umbc and umcp institutional animal care and use committees approved all procedures with animals and animal - derived materials . mdsc were plated in serum - free hl-1 medium ( biowhittaker , walkersville , md ) and maintained at 37 c with 5% co2 . after 18 h , the cultures were centrifuged at 805 g for 5 min ( eppendorf 5810 rotor , eppendorf , hamburg ) , the pellets were discarded , and the supernatants were centrifuged at 2090 g for 30 min ( sorvall rc5c , ss34 rotor , dupont , wilmington , de ) . the supernatants were then ultracentrifuged at 100 000 g for 20 h at 10 c ( beckman l8 , sw40ti rotor , beckman , pasadena , ca ) . the supernatants were discarded , and the pellets containing the exosomes were resuspended in pbs . exosomes were lysed in an optimized lysis buffer of 8 m urea in 50 mm ammonium bicarbonate with 50 m of deubiquitinase inhibitor pr-619 ( lifesensors , malvern , pa ) and 1% of a protease inhibitor cocktail ( sigma - aldrich , st . they were centrifuged at 14 000 g for 30 min with a 3 kda molecular weight cut off filter , and the supernatants were discarded . after lysis , the buffer was diluted to 0.8 m urea in 50 mm ammonium bicarbonate . protein content before and after immunoprecipitation was measured by the quick start bradford assay ( bio - rad , hercules , ca ) . ubiquitinated proteins were enriched using protein a - sepharose 4b beads ( invitrogen , carlsbad , ca ) that had been incubated with anti - ubiquitin antibody 3933 ( cell signaling technology , danvers , ma ) in a 1:600 dilution with rotation for 4 h at 4 c . excess antibody was removed from the beads by washing with 0.8 m urea in 50 mm ammonium bicarbonate and centrifuging three times at 3000 g for 2 min . one - hundred micrograms of exosome lysate was added to the sepharose bead slurry and incubated with rotation overnight at 4 c . the sepharose bead slurry was washed with 50 mm ammonium bicarbonate and centrifuged at 1000 g for 5 min to remove nonspecifically bound proteins . bound proteins were eluted by incubating the sepharose bead slurry in 0.2 m glycine , ph 2.6 , for 1 h at 4 c and collected via centrifugation at 13 000 g for 5 min . enriched fractions of ubiquitinated exosomal proteins were subsequently processed either by tryptic digestion in gel or in solution and immunoprecipitation of peptides with glycinylglycine - modified lysine residues . proteomic studies were conducted on exosomal proteins enriched for ubiquitin conjugates by immunoprecipitation . three biological replicates were resuspended in 2% sds , 5% -mercaptoethanal , and 62.5 mm tris hcl and reduced at 90 c for 5 min . the samples were then loaded onto 816% polyacrylamide gels ( bio - rad ) and subjected to electrophoresis for approximately 50 min at 200 v , 15 ma , and 50 w. the gels were stained using coomassie blue ( 40% methanol , 20% acetic acid , 0.1% m / v coomassie blue reagent 250 ; thermo scientific , san jose ca ) stain and then cut into 13 slices . after destaining , tryptic digestion was performed on each gel slice overnight at 37 c . the extracted tryptic peptides were resuspended in 0.1% formic acid for injection into the lc ms / ms . enriched fractions of ubiquitinated exosomal proteins from five biological replicates were frozen , lyophilized , and resuspended in 50 mm ammonium bicarbonate . proteins were reduced with 20 mm dithiothreitol for 30 min at 56 c and alkylated with 10 mm methylmethanethiosulfonate for 45 min . one microgram of trypsin was added to each fraction , and digestion was performed overnight at 37 c . as a positive control , a ubiquitin dimer linked with an isopeptide bond at k48 ( life sensors , malvern , pa ) peptides with glycinylglycine - modified lysine residues were enriched using protein a - sepharose 4b beads coupled to anti - diglycyl - lysine antibody gx41 ( millipore , billerica , ma ) using the same procedure as that with the anti - ubiquitin antibody , except the anti - diglycyl - lysine antibody was prepared at a 1:1000 dilution . the fractions of immunoprecipitated ubiquitinated proteins were added to the sepharose bead slurry and incubated with rotation overnight at 4 c . the unbound fraction was removed via centrifugation at 500 g for 5 min . the sepharose bead slurry was washed with 50 mm ammonium bicarbonate and centrifuged at 1000 g for 10 s to remove nonspecifically bound peptides . bound peptides were eluted by incubating the sepharose bead slurry in 0.2 m glycine , ph 2.6 , for 1 h at 4 c and collected via centrifugation at 13 000 g for 5 min . the elution was repeated , and the two elution fractions were combined . prior to lc ms / ms analysis , all fractions were desalted with c18 toptip spin columns ( glygen , columbia , md ) and resuspended in 100 l of 0.1% formic acid . all fractions were subjected to one - dimensional gel electrophoresis on an 816% criterion precast gel ( bio - rad ) at 200 v , 50 ma , and 15 w for 56 min , followed by transfer to a pvdf membrane ( emd millipore , billerica , ma ) at 100 v , 350 ma , and 35w for 1 h. free ubiquitin , polyubiquitin , and ubiquitinated proteins were detected by blotting with anti - ubiquitin antibody 3933 ( cell signaling technology ) followed by anti - mouse igg - hrp ( cell signaling technology ) . protein bands were visualized with an image lab system ( bio - rad , hercules , ca ) using the gel - doc program ( kodak molecular imaging systems ) and the supersignal west dura chemiluminescent substrate ( thermo fisher scientific , waltham , ma ) . exosomal histones were extracted using the epiquick total histone extraction kit ( epigentek , farmingdale , ny ) according to the manufacturer s instructions and analyzed for ubiquitination via western blotting with anti - ubiquitin antibody 3933 as previously described . ms / ms analyses were performed on a shimadzu prominence nano hplc ( shimadzu scientific instruments , columbia , md ) in - line with an ltq orbitrap xl ( thermo fisher scientific ) . a 10 l aliquot of tryptic peptides was injected onto an acclaim pepmap 300 c18 precolumn ( dionex , sunnyvale , ca ) followed by desalting with 10% solvent a ( 97.5% h2o , 2.5% can , and 0.1% formic acid ) for 20 min . peptides were fractionated on a c18 analytical column ( 150 0.15 mm , 300 , grace davidson discovery sciences , deerfield , il ) with a linear gradient increasing from 0 to 40% solvent b ( 97.5% acn , 2.5% h2o , and 0.1% formic acid ) in 85 min , followed by an increase from 40 to 85% solvent b in 20 min . precursor scans were acquired in the orbitrap with a resolution of 30 000 at m / z 400 . in each cycle , the nine most abundant ions were selected for fragmentation by collisional induced dissociation , and product ion scans were acquired in the ltq . a dynamic exclusion of 1 repeat count over 180 s was used . peptide and protein identifications were made by the peparml meta - search engine against the uniprot mouse database ( july 2014 ) . for the in - gel digestion , all peptide identifications were filtered at 10% spectral fdr , and proteins were required to be supported by at least 2 unshared peptides , bounding a protein fdr at 1% . for the two - step immunoaffinity enrichment of glycinylglycine - modified lysine - containing peptides , all peptide identifications were filtered at a 5% spectral fdr . fixed modifications listed methylthio modification of cysteine , and proteins with at least one peptide with a lysine residue tagged with glycinylglycine were considered to be ubiquitinated . when a protein identification was based on a single peptide from the double immunoaffinity workflow , subcellular location and function assignments of the identified proteins were made using the protein information resource go slim ( http://pir.georgetown.edu ) using uniprot gene ontology annotations ( july 2014 ) . western blotting was used to determine if mdsc - derived exosomes contain ubiquitinated proteins . a general ubiquitin antibody was used as well as antibodies that recognize k48 and k63 linkages in ubiquitin chains . figure 1 confirms the presence of ubiquitinated proteins , including proteins with k48- and k63-linked branched ubiquitins . consequently , mass spectrometry - based proteomic strategies were used to identify conjugated proteins . in - gel digestion of immunoprecipitated ubiquitin - conjugated proteins table 1 lists 16 ubiquitinated proteins identified from glycinylglycine - tagged peptides recovered from tryptic digestion in gel . table 2 lists 38 proteins identified from glycinylglycine - modified peptides isolated after tryptic digestion in solution . the experimental design , with immunoaffinity isolation of glycinylglycine - modified peptides , leads to several protein identifications that are each based on a single tagged peptide and thus are less reliable than those listed in table 1 . these are listed in table 2 , and annotated spectra are provided in supporting information . antibodies used are ( left ) anti - ubiquitin 3933 , ( middle ) anti - k-48-linked polyubiquitin , and ( right ) anti - k-63-linked polyubiquitin . indicates that the protein was also identified in the in - gel digestion . to report a protein as being ubiquitinated , reliable identification was required of at least one peptide containing a kgg residue . initial identifications were made by the peparml meta - search engine ( see experimental section ) , and all candidate tandem mass spectra were confirmed manually ( supporting information figure s1 ) . a combined total of 65 tryptic peptides containing modified lysines correspond to 50 ubiquitinated proteins ( supporting information table s1 ) . only 10 of the 50 proteins identified were previously reported in an mdsc - derived exosome lysate , demonstrating that enrichment for ubiquitinated proteins enabled the identification of low - abundance proteins in exosomes . as suggested by the western blots in figure 1 , several histones were also observed to be conjugated at multiple unique and nonoverlapping sites , and the pro - inflammatory high mobility group protein b1 ( hmg b1 ) was found to be ubiquitinated . the glycinylglycine - lysine sites identified experimentally were compared to ubiquitination sites predicted in silico by a ubiquitination prediction tool , ubiprober . of the 65 peptides identified , the glycinylglycine - lysine sites in 42 peptides are ubiquitination sites predicted with probabilities > 0.7 , ubiprober s confidence level . tables 1 and 2 also present the glycinylglycine - tagged peptides , where it can be seen that 15 of the 65 peptides have been formed by tryptic cleavage at gg - derivatized lysine residues . this unexpected tryptic cleavage has been observed previously by others , and a control experiment was also carried out to confirm its occurrence under the conditions of the present investigation . a commercial ubiquitin dimer linked with an isopeptide bond at k48 was subjected to tryptic digestion as described in the experimental section . both the expected peptide lifagkggqledgr and the irregular peptide lifagkgg were identified by tandem mass spectrometry in approximately a 3:1 ratio ( data not shown ) . several comparisons were made between the ubiquitinated proteins and a larger set of 412 proteins identified in an earlier study of mdsc exosome lysates . figure 2 presents a comparison of the uniprot - derived locations of exosome proteins referenced to parental mdsc and illustrates an increased percentage of nuclear proteins in the ubiquitinated cohort and significantly lower percentages of cytosolic and plasma membrane proteins . the ubiquitinated nuclear proteins include nine histones and isoforms as well as other nucleic acid binding proteins ( transcription factor a , mitochondrial , density regulated protein , transcription initiation factor tfiid subunit 3 , and protein bodl1 ) ( tables 1 and 2 ) . it should be noted that histones , especially linker histones such as the histone h1 family , have been reported to be located in the cytoplasm and cell surface as well as the nucleus and that several histones are already known to be ubiquitinated , e.g. , refs ( 3032 ) . the observation of ubiquitinated histones was confirmed by western blotting using anti - ubiquitin antibody 3933 on the histone fraction recovered using a total histone extraction kit ( see experimental section ) ( data not shown ) . figure 2 also indicates that 12 of the ubiquitinated proteins have no assigned cellular location . protein locations assigned to mdsc - derived exosomal lysate ( 412 proteins ) in gray and the ubiquitinated cohort ( 50 proteins ) in black . gene ontology annotations and the uniprot database were used to compare distributions of protein sizes ( without ubiquitin ) , grand average of hydropathicity ( gravy ) scores , and isoelectric points ( pi ) of the 50 ubiquitinated proteins identified . the intact masses of the proteins are somewhat evenly distributed between 11 and 327 kda ( excluding ubiquitination ) . the gravy scores and pi distributions are shown in figure 3 , panels a and b , respectively . although both sets of data illustrate a wide range of gravy scores ( 0.081 to 1.627 ) and pi values ( 4.72 to 11.71 ) , proteins that are ubiquitinated in mdsc - derived exosomes tend to cluster as hydrophilic and basic proteins . seventy - two percent of the proteins identified have a gravy score less than 0.5 , and 50% of the proteins have a pi greater than 9.00 . ( ubiquitin is not included in these calculations ; its monomer has a gravy score of 0.489 and a pi value of 6.56 . ) the bias toward a high ph is consistent with observations by chen and co - workers , who report a greater abundance of positively charged amino acids in ubiquitinated proteins . distribution of ( top ) grand average of hydropathicity score ( gravy ) and ( bottom ) isoelectric point of mdsc - derived exosomal lysate ( 412 proteins ) in gray and the ubiquitinated cohort ( 50 proteins ) in black . among the combined cohort of 50 proteins , 34 have not been previously reported to be ubiquitinated ( tables 1 and 2 ) . among these , sorting nexin 13 has been observed to participate in endosomal trafficking of ubiquitinated proteins . identification of two ubiquitinated keratins is consistent with the proposed role of protein aggregation in invagination , the initial step of exosome formation . other ubiquitinated proteins that are thought to play important roles in endosome and exosome formation include leucine zipper ef hand - containing transmembrane protein 1 ( letm1 ) and endoplasmin . although the functions of the ubiquitinated proteins are not known , these two unconjugated proteins participate in transporting and maintaining the high luminal concentration of ca required for optimal exocytosis of exosomes . on the basis of protein assay results , approximately 10% of the mdsc - derived exosome lysate comprises ubiquitinated proteins . tandem mass spectrometry coupled with immunoprecipitation has been used successfully to isolate and identify 50 ubiquitinated proteins from mdsc - derived exosomes and to determine their positions of conjugation . five of these are associated with formation of endosomes and exosomes , consistent with earlier proposals . the skew of pi values toward basicity in the conjugated cohort of exosomal proteins may contribute to their concentration and retention by the progressive acidification that has been documented along the endosomal pathway . the presence of ubiquitinated histones in these exosomes should be considered in the context of heightened interest in extracellular histones and their proinflammatory activity . hmg b1 is another pro - inflammatory mediator of particular interest since it was recently established as a driver of mdsc accumulation and suppressive potency . interestingly , s100 a8 and s100 a9 , exosomal pro - inflammatory proteins previously demonstrated to contribute to the bioactivity of mdsc , have not been identified with a glycinylglycine - lysine modification , even though they are quite abundant and readily identified in the exosome data sets .
we provide evidence at the molecular level that ubiquitinated proteins are present in exosomes shed by myeloid - derived suppressor cells ( mdsc ) . ubiquitin was selected as a post - translational modification of interest because it is known to play a determinant role in the endosomal trafficking that culminates in exosome release . enrichment was achieved by two immunoprecipitations , first at the protein level and subsequently at the peptide level . fifty ubiquitinated proteins were identified by tandem mass spectrometry filtering at a 5% spectral false discovery rate and using the conservative requirement that glycinylglycine - modified lysine residues were observed in tryptic peptides . thirty five of these proteins have not previously been reported to be ubiquitinated . the ubiquitinated cohort spans a range of protein sizes and favors basic pi values and hydrophobicity . five proteins associated with endosomal trafficking were identified as ubiquitinated , along with pro - inflammatory high mobility group protein b1 and proinflammatory histones .
You are an expert at summarizing long articles. Proceed to summarize the following text: chemistry - 2-hydroxy-3-methylamino naphthoquinoidal compounds 2 - 9 were prepared from lawsone by the classic mannich reaction , using the appropriate amines and formaldehyde in an alcohol - based solution ( fig . 1 ) as previously described ( leffler & hathaway 1948 , lagrota et al . 1988 , dos santos et al . 2000 , lima et al . 2002 ) . 2000 , lima et al . 2002 ) ( fig . 2 ) . ( tecoma ) and purified by a series of recrystallisation steps ( pinto et al . 1980 ) . nor - lapachol ( 11 ) was obtained using the hooker oxidation method ( fieser & fieser 1948 ) . iso - lapachol ( 12 ) and 2-hydroxy-3-methyl-1,4-naphthoquinone ( 13 ) were both synthesised using previously described techniques ( hooker 1936 ) . phenazine 14 was prepared from lapachol ( 10 ) through a two - step process ( da silva jnior et al . lapachol ( 10 ) was catalytically reduced and used to obtain compound 16 using the same reaction ( da silva jnior et al . the phenazine compounds 18 - 20 were prepared from c - allyl lawsone as previously described ( da silva jnior et al . 3 : lapachol ( 10 ) , nor - lapachol ( 11 ) , iso - lapachol ( 12 ) , phthiocol ( 13 ) and phenazine compounds 14 - 20 . cytotoxicity tests with human hepatocellular liver carcinoma cell line ( hepg2 ) - hepg2 ( att , usa ) , kindly donated by universidade nova de lisboa , portugal , and normal monkey kidney cells ( bgm ) cells , donated by the university of minas gerais , were cultured in rpmi-1640 medium ( sigma - aldrich , usa , ref 6504 ) , containing 40 mg / l gentamicin ( schering - plough , usa ) and 10% heat - inactivated foetal calf serum in 5% co2 at 37c ( gibco , usa ) . after reaching confluence , the cell monolayer was then trypsinised , washed with culture medium , aliquoted into a flat - bottomed 96-well plate ( corning , usa , ref 3595 ) at 5 10 cells / well and incubated for 18 h at 37c to allow cell adhesion . the test and control compounds were added at various concentrations ( 188 - 4,324 m ) and the cell monolayer was incubated for another 24 h. cytotoxicity was next evaluated as described ( coutinho et al . 2013 ) with the [ 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide ] ( mtt ) assay by adding 20 l of 5 mg / ml mtt ( molecular probes , usa ) per well ( denizot & lang 1986 ) . after 3 h of incubation , the supernatant was discarded , 100 l of dimethyl sulfoxide ( dmso ) ( sigma - aldrich ) were added per well and the optical density was measured ( spectramax 340pc384 , molecular devices , usa ) at 570 nm for the test conditions and 630 nm for the background wells . cell viability was expressed as a percentage of the absorbance of the untreated control cells subtracted from the appropriate background measurement . the minimum lethal dose of 50% cells ( mld50 ) was determined as previously described ( de madureira et al . 2002 ) and further used to calculate the selectivity index ( si ) of the active compounds , defined as the ratio of the mld50 to the ic50 ( bzivin et al . . continuous culture of p. falciparum and in vitro tests of drug activity - blood - stage parasites from the chloroquine - resistant and mefloquine - sensitive p. falciparum w2 clone kept as described ( andrade - neto et al . 2004a ) were maintained at 37c in human erythrocytes ( a ) in complete medium ( rpmi-1640 , supplemented with 10% blood group ahuman serum ) that was changed daily as previously described ( trager & jensen 1976 ) . the effects of the compounds were determined relative to control parasites kept in culture medium only ( rieckmann et al . ring - stage sorbitol - synchronised parasites ( lambros & vanderberg 1979 ) were used after adjusting the parasitaemia and haematocrit values as specified in each test at a volume of 180 l / well in 96-well microtitre plates ( corning , ref . the drug activity was measured using the anti - histidine - rich protein ii ( hrpii ) test ( noedl et al . 2002 ) . for the hrpii test , the parasites were adjusted to 0.05% parasitaemia and 1.5% haematocrit , placed in plates containing the drugs and incubated for 24 h under standard culture conditions . the contents of six wells ( containing no test drugs ) were harvested and frozen in microtubes for later use as background measurement . after 48 h of incubation , next , 100 l of the lysed cells from each well were added to a plate that had been pre - coated overnight at 4c with the primary anti - hrpii antibody ( mpfm-55a , icllab , usa ) for immunoassay . phosphate - buffered saline at ph 7.2 supplemented with tween 20 ( sigma - aldrich ) ( 0.05% ) and bovine serum albumin ( sigma - aldrich ) ( 4% ) was used to block the plates to ( avoid non - specific antibody binding ) for 3 h at room temperature ( rt ) . after 1 h at rt , the plates were washed and 100 l / well secondary antibody solution ( mpfg-55p icllab ) were added , followed by incubation with 3,3,5,5-tetramethylbenzidine chromogen cat . the reaction was stopped by adding 1 m sulphuric acid and the absorbance was read ( 450 nm ) using a spectrophotometer ( spectramax 340pc384 , molecular devices ) . falciparum drug activity was measured by comparing the drug concentrations that inhibited parasite growth to drug - free control cultures , which were representative of 100% cell growth . growth data from the serial drug dilutions to sigmoidal dose - response curves was analysed by curve - fitting software ( microcal origin software v.5.0 , originlab co , usa ) and used to measure inhibitory concentration for 50% ( ic50 ) of parasite growth . only compounds exhibiting ic50 values lower than 5 m were considered active ; those with ic50 values between 5 - 30 m were considered partially active and those with ic50 values higher than 30 m were considered inactive compounds . antimalarial tests against plasmodium berghei in mice - the antimalarial suppressive test was performed as previously described ( peters et al . briefly , p. berghei ( nk65 ) chloroquine - sensitive blood - stage parasites were stored at -70c and passaged weekly in outbred swiss mice . adult female mice weighing 20 2 g were inoculated with 1 10 infected erythrocytes , randomly distributed into groups of three mice per group after 24 h infection and then drug treated daily by gavage for three consecutive days . all compounds were freshly diluted in 3% dmso in rpmi medium and used at doses of 100 mg / kg ; chloroquine was used at 20 mg / kg and control mice received the drug vehicle . at days 5 and 9 after parasite inoculation , blood was taken from the tails of the mice and used to prepare smears , which were then fixed with methanol , giemsa - stained and examined microscopically to determine parasitaemia as previously described ( andrade - neto et al . the inhibition of parasite growth by drug treatment was evaluated in relation to the untreated mice , which were representative of 100% parasite growth . compounds that reduced parasitaemia by more than 40% were considered active , whereas those that reduced parasitaemia by 30 - 40% or less than 30% were classified as partially active and inactive , respectively . ethics - the protocols for animal use in the laboratory were previously approved by the ethical committee for animal use ( l-0046/08 ) , oswaldo cruz foundation , brazil . eight 2-hydroxy-3-methylamino naphthoquinones , seven phenazines and lapachol , nor - lapachol , iso - lapachol and phtiocol were evaluated for their in vitro activity against p. falciparum blood - stage parasites . the standard antimalarial chloroquine was tested as a control agent in parallel and the data are summarised in table i. table iactivity of quinones and phenazines against plasmodium falciparum inhibitory concentration for 50% ( ic50 ) measured through the anti - histidine - rich protein ii ( hrpii ) method , cytotoxicity [ minimum lethal dose of 50% cells ( mld50 ) ] against two different cell lines a human hepatoma ( hepg2 ) and a normal monkey kideny ( bgm ) and selectivity indexes ( si ) a ratio mdl50/ic50compoundsic50 hepg2bgm mld50 simld50 si21.3 0.4 3,363.12,564 3,363.12,564316.8 3.4 3,685.8220 3,685.8220414.1 7.0 3,886.7276 3,886.727651.9 0.72,075.1 316.51,0742,468.4 239.71,299630.6 5.7 4,324.4141 4,324.4141711.2 2.3331.1 105.329256.623859.9 7.7188.0 34.73321.5 28.35.49110.4 12.22,497.6 546.923 3,672.4331093 20,51,491.7 321.4161,785.1 888.3191126.9 0.91,698.6 94.8631,608.8 221.8601212.7 2.0 4,127.63241,388.9 592.510913 265.75,308.2 8.320 5,314.1201447.5 1.1 1,80338 1,8033815 94.21,041.0 58.511 1,884.6201620.6 2.6951.6 115.646632.8 11.431179.0 0.1 3,120.8348 - -1813.4 2.2 3,492.5260 3,492.52601960.5 9.2 2,005.933752 169.91220 99.5 1,989.8201,047.6 43.610chloroquine0.3 0.02460.4 97.31,535462.3 2.71,541 among the compounds tested , napthoquinones 2 and 5 were active , exhibiting ic50 values < 5 m ( 1.3 and 1.9 m , respectively ) . compounds 3 , 4 , 5 , 7 , nor - lapachol ( 11 ) and iso - lapachol ( 12 ) were considered partially active , as well as the phenazines 16 - 18 with ic50 within the range of 5 and 30 m . regarding cytotoxicity against both hepg2 and bgm cells , all molecules exhibited no toxicity based on high mld50 values observed . considering the therapeutic potential of the molecules examined , which was defined as the si based on the cytotoxicity against hepg2 cells and antiplasmodial activity in vitro , compounds 2 and 5 exhibited the highest si values of 2,564 and 1,074 , respectively , which were similar to or higher than that of chloroquine ( si = 1,535 ) . seven other molecules exhibited promising sis ranging from 100 - 350 ; these compounds were iso - lapachol ( 12 ) , three naphthoquinones derived from lawsone ( 3 , 4 , 6 ) and two phenazines ( 17 and 18 ) . nor - lapachol ( 11 ) , the naphthoquinone compound 7 , the phenazines prepared from lapachol ( 14 , 16 ) and phenazine ( 19 ) derived from c - allyl lawsone were less active ( si < 100 ) . other compounds displayed a low si ( 10 , 13 , 15 and 20 ) and were thus less promising ; one compound ( 8) exhibited an si value of 10 and was considered toxic . a near two - fold increase in cytotoxicity between hepg2 and bgm cells was observed for compounds 12 , 15 , 19 and 20 . to determine the in vivo activity of the selected compounds , 2 , 5 and 11 both compounds were found to be active by reducing parasitaemia by up to 63% , whereas nor - lapachol ( compound 11 ) was inactive in vivo ( table ii ) . analysis of the experimental compounds was performed once and in parallel to chloroquine , which served as a control antimalarial agent , clearing parasitaemia even at low doses ( 20 mg / kg ) and maintaining survival of the mice . the other treated animals succumbed to malaria infection with no delay in mortality caused by drug treatment with the new compounds . table iiantimalarial activity of nor - lapachol ( 11 ) and of the naphthoquinones 2 and 5 active in vitro , tested in mice with plasmodium berghei - malaria and orally treated with 100 mg / kg drug dose for three consecutive dayscompounds inhibition of parasitaemia at days after parasite inoculation ( % ) fifthninthactivity26341yes56348yes112121nochloroquine100100cure a : the experiment was performed in parallel with chloroquine , which cleared parasitaemia and allowed all mice to survive after treatment with a 20 mg / kg dose . a : the experiment was performed in parallel with chloroquine , which cleared parasitaemia and allowed all mice to survive after treatment with a 20 mg / kg dose . the synthetic strategy for the preparation of the 2-hydroxy-3-methylamino naphthoquinoidal derivatives ( 2 - 9 ) was used to obtain substances with aliphatic ( 5 - 8 ) and cyclic ( 2 - 4 and 9 ) substituents ( lima et al . the structures of these naphthoquinonoid compounds have been previously published , although our study is the first to report their potent activity against p. falciparum parasites . the synthetic methods described in this study were completed with commercial reactants and at high yields for quinoidal structures based on atovaquone . these compounds were prepared through a single step synthesis in contrast to the production of atovaquone , which is generally obtained in four steps . the phenazine compounds were easily prepared from lapachol derivatives and their activities have been described as antitumoural ( da silva jnior et al . our results showed that compounds 2 and 5 were active against p. falciparum in vitro , exhibiting high sis and promising therapeutic activity , similar to that of chloroquine . we considered the si an ideal parameter to evaluate new antimalarials , as this index reflects both drug toxicity and activity , thereby excluding toxic compounds from further study . using the si as a screening parameter investigations into the antimalarial activity of these compounds after structural modification are currently underway in our laboratories and will be reported in future work after establishing a structure - activity relationship . compounds 2 and 5 , which were highly active in vitro against p. falciparum , were also active against malaria in mice by inhibiting parasitaemia ; however , they could not prevent mortality from malaria . all compounds were tested at high doses because previous studies have indicated that lapachol has low activity in vitro ( carvalho et al . it is possible that these compounds possess low oral bioavailability , similar to most naphthoquinones ( ioset 2008 ) . in addition , slow uptake and/or rapid elimination of their active metabolites other phenazines derived from -lapachone partially active against p. falciparum in vitro were only active against malaria in mice when administered subcutaneously , but not orally ( andrade - neto et al . compounds 2 and 5 had a high therapeutic activity in vitro were tested only through oral administration in malaria - infected mice . the mechanism of quinone activity involves the generation of reactive oxygen species due to molecular structures that confer redox properties through reduction by one or two electrons . oxidative stress and/or the alkylation of lipids , proteins and nucleic acids by naphthoquinones may lead to cell damage ( vilamil - fernandez et al . 2004 ) , which accounts for their antimalarial ( dos santos et al . 2004 ) and antileukemic ( cavalcanti et al . 2013 ) activities . the ability to collapse mitochondrial membrane potential has also been described for atovaquone ( srivastava et al . 1997 ) and other naphthoquinones active against p. falciparum ( schuck et al . 2013 ) , a process that eventually leads to apoptosis or necrosis ( brand et al . whether the mechanism of action of these molecules is related to such processes remains to be determined . the cytotoxicity profiles were similar for all molecules using bgm or hepg2 cell lines . the two - fold differences observed for compounds 12 , 15 , 19 and 20 may be related to the distinct cellular processes exhibited by these two cells lines , which may have altered their sensitivity to the compounds . in conclusion , among the 19 compounds evaluated for anti - p . falciparum activity , the naphthoquinones derived from lawsone ( compounds 2 and 5 ) showed the highest activities with ic50 values < 5 m . they also showed the highest therapeutic effects which were similar to or higher than that of chloroquine . in addition , the cytotoxicities of the majority of the tested compounds were low . importantly , compounds 2 and 5 were shown to be active in vivo against malaria , causing a greater than 50% reduction of parasitaemia in mice infected with p. berghei . these compounds therefore show promise in the search for new antimalarial agents , with activities similar to that of atovaquone .
due to the recent advances of atovaquone , a naphthoquinone , through clinical trials as treatment for malarial infection , 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite plasmodium falciparum . these compounds include 2-hydroxy-3-methylamino naphthoquinones ( 2 - 9 ) , lapachol ( 10 ) , nor - lapachol ( 11 ) , iso - lapachol ( 12 ) , phthiocol ( 13 ) and phenazines ( 12 - 20 ) . their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines . compounds 2 and 5 showed the highest activity against p. falciparum chloroquine - resistant blood - stage parasites ( clone w2 ) , indicated by their low inhibitory concentration for 50% ( ic50 ) of parasite growth . the therapeutic potential of the active compounds was evaluated according to the selectivity index , which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro ic50 . naphthoquinones 2 and 5 , with activities similar to the reference antimalarial chloroquine , were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive . based on their in vitro and in vivo activities , compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study .
You are an expert at summarizing long articles. Proceed to summarize the following text: it is one of the most common diseases worldwide ( halbert et al 2006 ) and its incidence keeps growing along with the large diffusion of its main etiologic factor , tobacco smoking . the ever - increasing use of tobacco in the developing world allows for the prediction of a huge increase in the number of cases in the near and distant future . the prognosis of pulmonary emphysema is poor , with airflow limitation progressing to chronic respiratory insufficiency , disability and premature death . to this day , patients who stop smoking do benefit in terms of lung function and survival , but the decline in lung function is not reversed , merely slowed . apart from long - term oxygen therapy , pharmacological intervention is unsatisfactory and is mainly limited to the control of symptoms related to frequently - associated chronic bronchitis ( sutherland and cherniack 2004 ) . lung volume reduction surgery can be of interest in selected patients ( fishman et al 2003 ) as may be the implantation of endobronchial one - way valves ( wood et al 2007 ) but those techniques do not remedy the underlying condition . lung transplantation , while beneficial in terms of symptoms - related quality of life , does not improve survival in patients with pulmonary emphysema ( hosenpud et al 1998 ) . most studies of the pathogenesis of emphysema focused on the mechanisms involved in chronic injury induced by cigarette smoke to the pulmonary parenchyma . in particular , the role of protease - antiprotease imbalance , persistent inflammation , oxidative stress and excessive apoptosis of alveolar cells was demonstrated ( barnes et al 2003 ) . however , while great advances were made towards the comprehension of those phenomenons , they have not translated into new therapeutic prospects until now . on the one hand , alterations or defects in those systems may account for the deficiency in tissue repair which is observed in the emphysematous lung . on the other hand , the induction of alveolar regeneration would be an interesting therapeutic target in this disease . in this review , we will successively assess the evidence ( 1 ) that alveolar regeneration systems are functional in the mammalian lung and have the potential to regrow lost alveoli , ( 2 ) that cigarette smoke , the main etiologic factor of emphysema , inhibits those systems under experimental conditions , and ( 3 ) that alveolar regeneration systems are dysfunctional in the human emphysematous lung and may be a target for therapeutic intervention in this disease . the potential for alveolar generation and regeneration in adult mammals was only recently demonstrated in rodents but also in larger animals . this phenomenon could be observed after alveolar depletion was induced either diffusely by starvation or locally by the surgical resection of lung parenchyma . alveolar regeneration has been observed in mice fed a normal diet after a starvation period . starvation has long been known to induce the loss of pulmonary alveoli and the subsequent development of emphysema - like lesions in the lung as was recently shown in humans with anorexia nervosa ( coxson et al 2004 ) . there are no data regarding the potential reversibility of these lesions in humans , however the return to a normal diet induces the complete resolution of starvation - associated emphysema in mice ( massaro et al 2004 ) . this phenomenon represents a truly regenerative process as the restoration of normal numbers and surface of alveoli is accompanied by a sharp elevation of dna synthesis in the lung , indicating cellular replication ( massaro et al 2007 ) . indeed , the knowledge that the remaining lung undergoes adaptative changes and compensatory alveolar generation after lung resection was acquired several decades ago ( cagle and thurlbeck 1988 ) . in rats , resection of the upper and middle lobes of the right lung ( 25% of the total lung volume ) induces a sharp increase in the volume of the remaining lobes , while microscopically alveolar size and surface are close to those of unoperated animals , indicating the de novo generation of alveoli ( wandel et al 1983 ) . in mice , post - resection alveolar generation involves all cellular components of the lung as epithelial , endothelial and mesenchymal cells proliferate ( voswinckel et al 2004 ) . it must be emphasized that in this context the newly formed alveolar tissue seems to be functional and to participate in ventilation . of major interest , compensatory alveolar generation after lung resection arrested species , albeit to a lesser degree than in rodents . in dogs as well as in humans , the intensity of post - pneumonectomy alveolar generation seems to depend on age . in young dogs , after left pneumonectomy removing less than 45% of total lung parenchyma , compensatory alveolar generation occurs vigorously and leads to the restoration of alveolar numbers similar to that of unoperated animals ( takeda et al 1999 ) . by contrast , in fully adult dogs , a stronger stimulus such as right pneumonectomy ( removing 55% or more of parenchyma ) is needed to induce a weaker and incomplete response ( hsia et al 1994 ) . in humans , post - pneumonectomy alveolar generation and lung growth do not seem to occur in adults but indirect clues point towards the existence of such phenomenon in children and to a lesser degree in adolescents . in particular , the ventilatory function at adulthood of patients pneumonectomized before the age of 4 is close to that of unoperated subjects , most likely indicating that the formation of new alveoli compensated for resected ones ( laros and westermann 1987 ) . patients operated upon between 6 and 20 years of age still had better lung function parameters than those of patients who underwent pneumonectomy at adulthood ( laros and westermann 1987 ) . altogether , those results indicate that alveolar regeneration systems are present in the lung of growth - arrested mammals such as humans , and that those systems are accessible to stimulation . alveolar cells of every type ( epithelial , endothelial , and mesenchymal ) participate in alveolar multiplication and regeneration events since those require their equilibrated proliferation . moreover , all three main cell types seem to possess the ability to drive lung growth since the administration of growth factors targeted towards either type leads to the acceleration of alveolar generation after lung resection . the hepatocyte growth factor ( hgf ) , the first such agent that was identified as stimulating lung growth after lung resection ( yanagita et al 1993 ) , targets both epithelial and endothelial cells . the epidermal growth factor ( egf ) ( kaza et al 2000 ) , activates mainly epithelial cells , whereas the platelet derived growth factor - bb ( pdgf - bb ) ( yuan et al 2002 ) is a mitogen primarily for mesenchymal cells . while both the keratinocyte growth factor ( kgf ) ( kaza et al 2002 ) , and the vascular endothelial growth factor ( vegf ) ( sakurai et al 2006 ) stimulate post - pneumonectomy alveolar multiplication , the former induces proliferation exclusively in epithelial cells while the latter essentially targets the endothelium . altogether , those results suggest that the induction of proliferation in a particular cell population of alveoli may be accompanied by the simultaneous expansion of the other neighboring cell types , possibly through paracrine mechanisms . however , some elements indicate that mesenchymal cells may be first among equals and may play a leading part in those processes . indeed , pulmonary fibroblasts are the main contributors to the synthesis and maintenance of the alveolar extracellular matrix which is the essential scaffold over which other cells proliferate and differentiate and which provides the lung with its essential mechanical characteristic , elasticity . pulmonary fibroblasts are the main cell type responsible for the synthesis and secretion of the main components of the alveolar extracellular matrix , proteoglycan , collagen and elastin . elastin in particular is an essential component of the alveolar extracellular matrix as it provides the lung with the elasticity needed for ventilation , and it is probable that elastin synthesis is an essential step in alveolar growth and regeneration . supporting this hypothesis , koh and colleagues ( koh et al 1996 ) showed that tropoelastin mrna was sharply overexpressed in the lung of rats after pneumonectomy , and that its expression was localized to the ends , bends and junctions of alveolar walls , indicating that the de novo synthesis of elastic fibers was part of the process of alveolar growth and multiplication . in this study , the localization of lung elastin mrna expression after pneumonectomy differed slightly from that observed in developing animals as in the latter case elastin mrna expression was observed at the end and bend of alveolar walls but was not observed at the junctions of alveolar walls . in addition to their role as extracellular matrix providers , alveolar fibroblasts seem to participate in the complex cellular interactions that lead to alveolar growth and multiplication both in the fetal lung ( warburton and bellusci 2004 ) and in the adult lung . fibroblasts interact both with endothelial cells and epithelial cells through the secretion of soluble factors that act in a paracrine fashion . in particular , fibroblasts are the main if not the only source of kgf in the distal lung and contribute greatly to the elevation of lung hgf levels in response to alveolar injury ( stern et al 2000 ; marchand - adam et al 2003 , 2005 ; cohen et al 2006 ) . moreover , although this point has not been demonstrated in adult animals so far , lipid - laden interstitial fibroblasts , a subpopulation of alveolar fibroblasts , are the main source of retinoic acid in the lungs of rodents undergoing alveolar septation ( mcgowan and torday 1997 ) . in adult animals , retinoic acid , the active metabolite of vitamin a , increases post - pneumonectomy lung growth in rodents ( kaza et al 2001 ) and enhances alveolar capillary formation after right pneumonectomy in dogs ( yan et al 2004 ) . interestingly , the role of fibroblasts in the regulation of cell - cell interactions in alveoli may not be limited to the secretion of paracrine signal molecules as cytoplasmic expansions originating in those cells reach alveolar endothelial and epithelial cells through their respective underlying basement membranes ( sirianni et al 2003 ) . overall , fibroblasts seem to play a central role in alveolar multiplication and regeneration and it may be assumed that the realization of such phenomenon requires proliferating and metabolically active fibroblasts . the mechanisms by which chronic smoke exposure induces chronic lung injury leading to emphysema have been exhaustively described and include chronic inflammation ( hoidal and niewoehner 1982 ) , protease / antiprotease imbalance ( carp and janoff 1978 ) , oxidative stress ( church and pryor 1985 ) and excessive death of bronchiolar and alveolar epithelial and endothelial cells ( jung et al 2000 ; tuder et al 2000 ; wickenden et al 2003 ) . however , the role of cigarette smoke in the pathogenesis of emphysema does not seem to be limited to its participation in chronic injury to the lung . indeed , cigarette smoke has been shown to reduce viability in human lung fibroblasts and to inhibit a number of fibroblast functions closely linked to alveolar regeneration , at least in vitro . cigarette smoke possesses cytotoxic properties towards lung fibroblasts as cigarette smoke extract reduces the viability of those cells in vitro ( ishii et al 2001 ) . this effect has been linked to the induction of apoptosis ( carnevali et al 2003 ; baglole et al 2006 ) . additionally , cigarette smoke inhibits lung fibroblast proliferation and their capacity to migrate ( nakamura et al 1995 ; nobukuni et al 2002 ) . moreover , lung fibroblasts chronically exposed to cigarette smoke display a senescent phenotype characterized by an enlarged morphology and cell cycle arrest ( nyunoya et al 2006 ) . altogether , those results indicate that cigarette smoke probably induces a reduction in the number of metabolically active fibroblasts available for alveolar growth and multiplication in the emphysematous lung . in addition to its cytotoxic and antiproliferative effects , cigarette smoke inhibits key fibroblastic functions associated with alveolar growth and multiplication . in particular , elastin synthesis and cross - linking seem to be especially sensitive to exposure to cigarette smoke . while no data are available regarding adult lung fibroblasts , cigarette smoke extract down regulates tropoelastin mrna in rat fetal lung fibroblasts ( gao et al 2005 ) . in an acellular model , cigarette smoke inhibits elastin cross - linking ( laurent et al 1983 ) , while the transcription of lysyl oxidase , a key effector of elastin cross - linking is reduced in fetal rat lung fibroblasts exposed to cigarette smoke extract ( gao et al 2005 ) . whether cigarette smoke exposure antagonizes alveolar growth and multiplication in vivo has not been demonstrated directly . to our knowledge , no study has been dedicated to the description of the effect of cigarette smoke on post - pneumonectomy lung growth . however , data obtained in a different model of alveolar depletion , elastase - induced emphysema , indicate that cigarette smoke exposure may indeed induce a defect in elastin synthesis and cross - linking , alveolar growth , multiplication and/or repair . indeed , while elastase instillation in the trachea induces a sharp elevation in elastin synthesis in the lung , this response is reduced by 40% in the lung of hamsters exposed to cigarette smoke for one week ( osman et al 1985 ) . this reduction in elastin synthesis is accompanied by a seven - fold reduction in lysyl oxidase activity in the lung of those animals . this reduction in elastin synthesis and cross - linking may be of great pathophysiological significance as emphysema lesions are exaggerated in hamsters exposed to cigarette smoke after elastase instillation ( hoidal and niewoehner 1983 ) . whether a defect in alveolar regeneration systems plays a role in cigarette smoke induced emphysema in humans remains unclear but some elements indicate that such systems do get activated in the course of this disease . for instance , contrary to what is observed in the normal lung ( shapiro et al 1991 ) , a high elastin turnover is observed in the emphysematous lung ( stone et al 1995 ; gottlieb et al 1996 ; vlahovic et al 1999 ) , indicating that in parallel with the excessive degradation of elastic fibers which is observed in this disease , elastin synthesis occurs in the lung . interestingly , in line with a high rate of extracellular matrix synthesis associated with a high rate of extracellular matrix degradation , a high rate of cellular proliferation is associated with the elevated apoptosis of alveolar cells in the emphysematous lung ( imai et al 2005 ) , adding more substance to the hypothesis that alveolar regeneration systems do get activated in the emphysematous lung even though they are probably not functional as the alveolar architecture is not restored . while an alteration of alveolar regeneration systems has not been directly evidenced in the emphysematous lung ( bonay et al 2005 ) , it must be put forward that alveolar fibroblasts exhibit a defective phenotype in the emphysematous lung . available data regarding the in situ phenotype of pulmonary fibroblasts in emphysema are limited to the observations by sirianni and coworkers ( sirianni et al 2006 ) . these authors showed that direct intercellular contacts between alveolar fibroblasts and alveolar epithelial cells or capillary endothelial cells are greatly reduced in the emphysematous lung . those results suggest that pulmonary emphysema fibroblasts may lack the capacity to participate in the direct intercellular communications which play an essential role in fetal lung development and probably during lung repair in the lung ( warburton et al 2001 ; parker et al 2004 ; warburton and bellusci 2004 ) . the abnormal phenotype of alveolar fibroblasts in pulmonary emphysema was described more extensively in vitro on primary lines of pulmonary fibroblasts obtained following the explant culture technique . first , pulmonary emphysema fibroblasts show signs of premature senescence . when compared to fibroblasts obtained from normal human lung , fibroblasts obtained from emphysematous lung have a markedly reduced proliferation rate . indeed , emphysema fibroblast proliferation was described as 50%60% of controls ( nobukuni et al 2002 ; noordhoek et al 2003 ; holz et al 2004 ) . in one study , those cells were shown to express senescence - associated beta - galactosidase ( sa - beta - gal ) , a marker of cellular senescence ( muller et al 2006 ) . moreover , in some studies emphysema fibroblasts did not exhibit the typical spindle - shaped microscopic appearance of fibroblasts and expressed smooth - muscle actin , indicating a degree of differentiation towards the myofibroblast phenotype . secondly , in addition to premature senescence of pulmonary emphysema fibroblasts precluding an elevation of their number , those cells seem to secrete low levels of growth factors . in particular , hgf secretion by those cells is sharply reduced compared to normal lung fibroblasts and they poorly respond to exogenously added kgf ( plantier et al 2005 ) . given the potential regenerative effect of hgf and kgf in the lung , a low secretion of this factor by lung fibroblasts may play a role in the pathogenesis of emphysema . altogether , those results indicate an altered phenotype of pulmonary fibroblasts which may partly contribute to the absence of alveolar regeneration observed in emphysema . the therapeutic manipulation of alveolar regeneration systems would represent a tremendous advance for the treatment of emphysema as well as other diseases characterized by the destruction of alveoli . such results were obtained in animal models of emphysema through the administration of pharmacological agents , gene therapy or the implantation of mesenchymal stem cells , raising hope that such an effect may be attainable in human patients . the proof of concept that alveolar regeneration was a possible therapeutical goal in emphysema was brought by the pioneering work by donald and gloria massaro ( massaro and massaro 1997 ) . in this study , all - trans retinoic acid induced partial alveolar regeneration 20 days after emphysema was induced in rats with the tracheal instillation of elastase . this result was reproduced by some groups ( ishizawa et al 2004 ) , while others did not ( tepper et al 2000 ; lucey et al 2003 ; fujita et al 2004 ; march et al 2004 ) . the strong regenerative effect of exogenous hgf was shown in the elastase - induced emphysema model in mice . its effect was associated to the engraftment into the lung of cells derived from the circulation ( ishizawa et al 2004 ) . this effect was reproduced in rats transfected intravenously with the human hgf gene seven days after the instillation of elastase ( shigemura et al 2005 ) . adrenomedullin , an angiogenic factor , has been shown to possess alveolar regenerative properties in the same model , albeit at a lesser degree than hgf ( murakami et al 2005 ) . interestingly , an angiogenic stimulus seems necessary for the induction of alveolar regeneration in the emphysematous lung as kgf , which targets mainly epithelial cells , was not found to exert such an effect , even though its cytoprotective properties allowed it to fully prevent the constitution of emphysema in mice instilled with elastase ( plantier et al 2006 ) . all of the studies cited so far relied on the administration of soluble factors , either directly or by gene transfection . another approach to the development of alveolar regenerative therapeutics has been the transplantation into the lung of mesenchymal stem cells obtained from adipose tissue ( shigemura et al 2006a , 2006b ) . adipose - tissue derived stromal cells were obtained from inguinal subcutaneous fat and applicated to the elastase - injured lung , resulting in alveolar regeneration and higher levels of pao2 and maximal oxygen consumption . their regenerative effect in the emphysematous lung may be related to this characteristic as their engraftment into the lung was not reported . overall , the feasibility of inducing alveolar regeneration in rodents with elastase - induced emphysema has been thoroughly demonstrated . whether alveolar regeneration may be obtained in pulmonary emphysema patients in the near future remains unsure . to this day , a single study has been devoted to the exploration of the effect of a regenerative agent in this disease , namely retinoic acid ( roth et al 2006 ) . in this crossover study , all - trans retinoic acid , 13-cis retinoic acid or placebo were administered for 6 months to 148 patients with moderate - to - severe copd and a predominant component of emphysema . despite the fact that high plasma levels of retinoic acid were obtained in treated patients , no difference in pulmonary function or quality of life was observed between groups in this study . among the candidate drugs for regeneration therapy in the lung , hgf seems to be of particular interest . while the use of this factor for the treatment of lung disease has not been reported to date , encouraging preliminary results have been obtained in the treatment of peripheral arterial disease where hgf is well tolerated in the short term and induces an increase in distal perfusion ( morishita et al 2004 ) . however , hgf has been reported to facilitate the growth of tumors in various organs , including the lung ( stabile et al 2006 ) . since patients with pulmonary emphysema are most frequently smokers or ex - smokers at a high risk of lung cancer , whether hgf itself is a good candidate for the long - term treatment of this disease remains to be evaluated . alveolar regeneration can be induced in animal models of diffuse or localized alveolar loss and can be repressed by exposure to cigarette smoke . defects in alveolar regeneration systems have been identified in the human emphysematous lung and may play an important role in the course of this disease which is characterized by alveolar loss . restoration and/or induction of these systems in emphysematous patients may represent a major advance in the treatment of pulmonary emphysema , a disease characterized by the loss of pulmonary alveoli . encouraging results have been obtained in elastase - induced emphysema in rodents , and agents known to induce alveolar regeneration in this model are currently under evaluation for the treatment of emphysema and other diseases .
pulmonary emphysema is characterized by the irreversible loss of pulmonary alveoli . despite recent advances in the understanding this disease , its treatment remains palliative . in this review , we will successively review the data suggesting ( 1 ) that alveolar regeneration systems are functional in the mammalian lung and have the potential to regrow lost alveoli , ( 2 ) that cigarette smoke , the main etiologic factor of emphysema , inhibits those systems under experimental conditions , and ( 3 ) that alveolar regeneration systems are dysfunctional in the human emphysematous lung and may be a target for therapeutic intervention in this disease . special emphasis will be put on the role of alveolar fibroblasts in those processes .
You are an expert at summarizing long articles. Proceed to summarize the following text: it was shown long ago in experimental infection of mice that passive transfer of polyclonal antibody specific to influenza a haemagglutinin ( ha ) very efficiently protects animals infected intranasally ( in ) with a virulent strain of influenza virus ( a / pr8/33 ) . the main conclusions from this original publication were as follows : protection was complete , as all animals could be protected against in inoculation able to kill 100% of control mice ( inoculated with irrelevant rabbit serum).protection strictly correlated with profoundly diminished virus replication , and with decreased lung consolidation.protection could be afforded by antibody transfer even when transfer was delayed by 48 h , thus occurring after completion of the first cycles of virus replication ( deaths were observed between 7 and 12 days in untreated controls).protection could be obtained by transfer of relatively low amounts of rabbit antiserum to homologous ha , 100fold lower than the antiserum amount providing detectable levels of transferred serum antibody , as measured in a singleradial diffusion test performed 1 h after antiserum injection.igg class antibodies circulating systemically were found to be sufficient for protection against this mucosal infection . protection was complete , as all animals could be protected against in inoculation able to kill 100% of control mice protection could be afforded by antibody transfer even when transfer was delayed by 48 h , thus occurring after completion of the first cycles of virus replication ( deaths were observed between 7 and 12 days in untreated controls ) . protection could be obtained by transfer of relatively low amounts of rabbit antiserum to homologous ha , 100fold lower than the antiserum amount providing detectable levels of transferred serum antibody , as measured in a singleradial diffusion test performed 1 h after antiserum injection . igg class antibodies circulating systemically were found to be sufficient for protection against this mucosal infection . in the mouse experimental model described above , the specificity of the antibody transferred was shown to be critical : protection was obtained with antisera to the ha homologous to the virus used for challenge ( a / pr8/33 ) , but not antisera to the ha of a strain ( a / fm1 ) having drifted antigenically within the h1 subtype.this indicated that only antibodies to strainspecific determinants of ha are protective , a major finding which appears to account for the basic strategy of escape of influenza virus , i.e. a subtle , yet critical antigenic drift within a given subtype , permitting the succession , in human populations , of several epidemics with variant strains of the same subtype virus.thus very few changes in ha sequences are needed to bypass immune memory and permit escape from the protective effects of antibodies induced in human populations by the previous epidemic variant . better still , such modest changes in sequence remain compatible with a good viral fitness for replication and transmission , another critical condition needed for any virus to maintain its epidemic potential . protection was obtained with antisera to the ha homologous to the virus used for challenge ( a / pr8/33 ) , but not antisera to the ha of a strain ( a / fm1 ) having drifted antigenically within the h1 subtype . this indicated that only antibodies to strainspecific determinants of ha are protective , a major finding which appears to account for the basic strategy of escape of influenza virus , i.e. a subtle , yet critical antigenic drift within a given subtype , permitting the succession , in human populations , of several epidemics with variant strains of the same subtype virus . thus very few changes in ha sequences are needed to bypass immune memory and permit escape from the protective effects of antibodies induced in human populations by the previous epidemic variant . better still , such modest changes in sequence remain compatible with a good viral fitness for replication and transmission , another critical condition needed for any virus to maintain its epidemic potential . antibodies to ha are by far the most protective providing that there is a good match to the specificity of ha determinants present in the distal ( ha1 ) portion of the molecule . this situation is exploited strategically by the influenza virus to remain epidemic on the planet for tens of years , through subtle antigenic drift . the need to maintain a high level of replication fitness is likely not to be compatible with most random ha antigenic changes . periodically , influenza virus must shift ha altogether , by exchanging ( reassorting ) its ha genome segment with that of another influenza strain from the varied genome repertoire of the avian flu reservoir , or create a new pandemic virus by adaptation to man of a pathogenic , avian virus with an ha however , protection is provided in a matter of a few days , in each infected individual , by the early production of antibody to the novel strainspecific determinants encountered . the few days needed for such antibodymediated virus eradication in a given individual is nevertheless sufficient for efficient , aerosolmediated dissemination of the virus to other susceptible individuals , thus permitting persistence of the epidemics . the design of antibodybased immune intervention against influenza infection ( whether based on active immunization through vaccine , or on passive transfer of antibodies ) must thus target either ha determinants as close as possible to those of the infecting virus , or determinants conserved among many flu strains over times . heterosubtypic immunity ( hsi ) to influenza , i.e. protective crossreactivity to lethal infection with influenza a virus of a serotype different from the virus initially encountered . it was expected that protection would be afforded by cytotoxic lymphocytes , known to respond preferentially ( in an contrary to expectation however , it was found that mice deficient in cd8 molecules performed hsi normally , whereas b celldeficient animals did not . this and other experiments suggested that heterotypic protection within an influenza subtype relies on humoral , but not cd8mediated t cell immunity . clearly , the specificity of the b memory cells involved and that of the corresponding antibodies must be to antigens conserved within a subtype . the influenza virus genome segment coding for the neuraminidase protein is less variable than the ha segment . in addition to the major role played by antibodies to ha , some protection can indeed be transferred by antibody to influenza neuraminidase ( na ) . this was shown by passive transfer experiments in mice , and was also indicated by epidemiological data in human populations . vaccines based on na were reported to be protective . to protect people against flu after an antigenic shift in both na and ha , it would of course be useful to target an antigen with very high conservation and expressed on the virus membrane . a very minor , yet potentially important , viral antigen present on the virion surface is the outer part of the transmembrane m2 antigen ( m2e ) , which is largely invariant . interestingly , it was shown that transfer of monoclonal antibody to m2 reduces the growth of both h1n1 and h3n2 viruses . however , the possibility of basing a universal influenza vaccine on immunization with m2e should be discussed with prudence , as it was shown that influenza mutants emerge in vivo in the presence of antibodies to m2e . the contribution of bird influenza under normal conditions is to provide a reservoir of 16 different ha ( and other viral genes ) , that influenza virus circulating in human populations uses periodically to undergo antigenic shift through genome reassortment , thus totally escaping protection by specific antibody for the entirely different ha present in the flu variants responsible for previous epidemics . however , it appears that from time to time that avian flu strains can become pathogenic to man , for molecular reasons not yet fully elucidated . it is likely , in particular , that mutations in some nonstructural proteins enable the virus to escape the protective effects of interferon which , together with antibody to ha , is a major host defence mechanism against flu infection . another major parameter of influenza adaptation of bird strains to human respiratory tract is modifications at the level of ha domains interacting with the sialic acid residues recognized on cell membrane proteins and lipids , used as receptors for virus entry . this adaptation may permit avian strains to recognize , instead of the avian 23 sialic acid receptors , the 26 type sialidation expressed by the human upper respiratory tract mucosal cells . in addition , the study of the spanish flu genome indicated that some mutations in the influenza polymerase gene increase the efficiency of the enzyme in vitro and the replicative efficiency of the virus in animal models . strains of avian flu with pathogenicity to humans were observed in asia during the last years . at the present stage , these strains remain poorly infective to man , in that a massive exposure ( for example in poultryassociated professions ) is needed for infection to occur . only few human cases are expected to be observed if these avian influenza strains do not mutate and do not acquire the capacity to be transmitted from man to man . however , the latter situation , if it ever happened , would provide conditions for a pandemic with a potential for morbidity and mortality rates comparable to the deadly spanish flu pandemic of 1918 , now suggested to have been caused by direct passage to man of an influenza virus strain with avian characteristics . thus strong evidence supports the concept that transfer of specific antibodies could efficiently protect people who were recently in close contact , professionally or not , with influenza virusinfected patients . other therapeutic interventions can be used , such as the taking of antiviral drugs , particularly drugs able to inhibit neuraminidase activity . however , it should be stressed that strains resistant to presently available antineuraminidase drugs have been reported . moreover , it was shown that these drugs are efficient only when taken within a few hours after exposure . interestingly , observations in the few people who were infected with the h5n1 bird virus indicate that the incubation period is relatively long , a delay which would give time to administer antibodies to h5 , with hopefully protective effects obtained even after the first virus replication cycles , as may be extrapolated from the experiments in mice described above . , of further interest is the observation that the h5n1 virus can be found in the digestive tract at autopsy , which provides further incentive to treat contaminated people systemically with highly diffusible igg antibodies to h5n1 virus . the evidence described and discussed in the present article strongly support the concept of designing and producing monoclonal antibodies aiming at protecting people recently exposed to highly pathogenic influenza virus . it is important to note that therapeutic transfer of antibody should be protective even against highly pathogenic influenza viruses . indeed , recent evidence indicates that mice immunized against the highly virulent 1918 spanish flu are efficiently protected against lethal challenge . this strong protection is not broken by tlymphocyte depletion , but can be transferred by igg from protected mice . this is in keeping with recent suggestions that polyclonal antibodies from convalescent people could have an important preventative effect on severe influenza infection , as indicated in a metaanalysis of pneumonia in the spanish influenza pandemic . recently reported that whereas circulating igg antibodies do protect against disease , they do not completely prevent infection of the nose . if these observations were valid in people , they would be compatible with the idea that transferred antibody would protect against flu disease but might not prevent some transmission of the virus in the population in contact with the individuals treated . although this may appear to be a limitation of the therapeutic approach consisting in transferring protective igg to influenza virus , that phenomenon may in fact have important , positive consequences . indeed , the giving of igg antibodies might actually act as a surrogate vaccine allowing those who would get infected despite antibody transfer to become immune without becoming sick . in addition to financial aspects , which are beyond the scope of the present , short opinion paper , numerous questions remain to be discussed about the technical aspects of the approach discussed here . main questions would concern : the route of administration , with the probability that subcutaneous injection will be efficient , and more readily accepted by people than intravenous administration.the nature of the mab to be used : igg mab of human origin ( rather than secondary humanization of animal mabs ) should be used . the biotechnology for large scale production of human immunoglobulins in different systems , including plant technology , has enormously progressed in these last years , ensuring a reasonable feasibility for the development phase . if plant technology was used , the lack of animal virus or prion contaminants would facilitate the acceptance of administration in humans by regulatory authorities.the specificity of the mabs to be raised : ha ( h5 if h5n1 virus were to be treated ) would obviously be a priority.however , administration of antibodies with more than one specificity in the same injection(s ) should be considered : the evidence reviewed above points to the potential interest of including ( in addition to the antiha mab ) other mabs , in particular those specific for the neuraminidase of the pandemic virus , or even to m2e . synergism in terms of protection , and prevention of antibodyinduced resistance to neutralization would be expected.the subspecificity and protective capacity of the antibody preparation to the pathogenic avian strain targeted should be screened by studies of its neutralizing activity in ovo and/or in cell cultures , and its protective efficiency verified in animal models , as performed by kong et al . in the case of the 1918 virus in mice . if , as suggested above , more than one mab was present in the preparation to be administered , the likelihood of obtaining a protective effect on influenza strains despite mutations occurring during the pandemic dissemination of the virus should remain very high . the route of administration , with the probability that subcutaneous injection will be efficient , and more readily accepted by people than intravenous administration . the nature of the mab to be used : igg mab of human origin ( rather than secondary humanization of animal mabs ) should be used . the biotechnology for large scale production of human immunoglobulins in different systems , including plant technology , has enormously progressed in these last years , ensuring a reasonable feasibility for the development phase . if plant technology was used , the lack of animal virus or prion contaminants would facilitate the acceptance of administration in humans by regulatory authorities . the specificity of the mabs to be raised : ha ( h5 if h5n1 virus were to be treated ) would obviously be a priority . however , administration of antibodies with more than one specificity in the same injection(s ) should be considered : the evidence reviewed above points to the potential interest of including ( in addition to the antiha mab ) other mabs , in particular those specific for the neuraminidase of the pandemic virus , or even to m2e . synergism in terms of protection , and prevention of antibodyinduced resistance to neutralization would be expected . the subspecificity and protective capacity of the antibody preparation to the pathogenic avian strain targeted should be screened by studies of its neutralizing activity in ovo and/or in cell cultures , and its protective efficiency verified in animal models , as performed by kong et al . in the case of the 1918 virus in mice . if , as suggested above , more than one mab was present in the preparation to be administered , the likelihood of obtaining a protective effect on influenza strains despite mutations occurring during the pandemic dissemination of the virus should remain very high .
the fear of a potential pandemic with a highly pathogenic influenza a virus , such as the avian virus h5n1 , has rightly prompted multidisciplinary reflections and calls for better preparedness all over the world . in terms of therapeutic aspects , most of the focus has been on vaccines and antivirals . the present opinion paper intends to discuss a different therapeutic approach , although not mutually exclusive to the two others quoted above . we here propose an approach , based on welldocumented experimental evidence in animal models , in which a short series of human monoclonal antibodies adapted to the probable pandemic strain , specific for external antigens of that influenza virus and shown in vitro and in experimental models to have neutralizing properties , are prepared and stockpiled for administration to people recently exposed to the pandemic virus .
You are an expert at summarizing long articles. Proceed to summarize the following text: mortality due to cardiovascular disease ( cvd ) has declined worldwide during the past few decades ( wijeysundera et al . , 2010 ) . however , during the same time period , the number of cvd deaths attributable to diabetes has increased ( puri et al . , 2012 ) . first , although the prevalence of other risk factors , such as smoking , hypertension and hypercholesterolemia , has been reduced by prevention programs , the incidence of diabetes has been steadily rising ( chen et al . , 2011 ) . second , there has been no significant decline in the increased cardiovascular risk experienced by diabetic subjects . clearly , there is an urgent need to curb the current epidemic of diabetes and to prevent cvd in diabetics . although little is known about the factors underlying the increased cardiovascular risk in diabetic patients , studies in diabetic and nondiabetic subjects suggest that the risk of cvd is influenced by genetic factors ( lusis et al . , 2004 ) . osteoprotegerin ( opg ) , also known as osteoclasto - genesis inhibitory factor , is a member of the tumor necrosis factor receptor superfamily of cytokines ( simonet et al . , clinically , opg may play a role in the development of osteoporosis , inflammatory bone diseases , multiple myeloma and malignant bone resorption ( goranova - marinova et al . , 2007 ; turk et al . , 2009 ) . in addition , opg has been associated with the presence and severity of cardiovascular events : elevated serum opg concentrations have been found to correlate with the severity of peripheral artery disease and heart failure , symptomatic carotid stenosis , unstable angina , vulnerable carotid plaques and acute myocardial infarction ( golledge et al . , 2004 ; crisafulli et al . , 2005 ; ueland et al . , 2005 ; ziegler et al . , 2005 ; sandberg et al . , 2006 ; kadoglou et al . , the gene encoding opg is affected by common , functionally important genetic polymorphisms that have been associated with osteoporosis and neuroarthropathy and are considered early predictors of cardiovascular disease ( collin - osdoby , 2004 ; soufi et al . , 2004 ; styrkarsdottir et al . , 2008 ; pitocco et al . , some recent studies have shown that the t245 g , t950c and g1181c polymorphisms of the opg gene are associated with the vulnerability of carotid plaques and risk of stroke ( straface et al . , 2011 ; biscetti et al . , 2012 ) . however , the extent to which these genetic markers predispose to increased cardiovascular complications in type 2 diabetes remains uncertain . the purpose of the present case - control study was therefore to determine whether the t245 g ( rs3134069 ) , t950c ( rs2073617 ) and g1181c ( rs2073618 ) polymorphisms of the opg gene play a role in cvd in type 2 diabetic patients . all participants were randomly recruited from the department of internal medicine of the first affiliated hospital of xinxiang medical university in weihui ( henan province , people s republic of china ) , from september 12 , 2007 , to july 20 , 2012 . type 2 diabetes was diagnosed according to who criteria ( alberti and zimmet , 1998 ) . age at diagnosis of type 2 diabetes was > 40 years in 98% of the subjects and the minimum age at onset was 37 years . type 1 diabetes was carefully excluded on clinical grounds based on a review of medical records , on the fasting c - peptide levels and on the absence of islet - related auto - antibodies . for the purpose of this study , cvd was defined as the occurrence of a fatal or nonfatal myocardial infarction or coronary artery bypass grafting . subjects diagnosed with cvd before the diagnosis of type 2 diabetes were excluded , as were those diagnosed with stroke and/or angina . after these exclusions , 181 women ( 117 cvd case subjects and 64 control subjects ) and 309 men ( 195 cvd case subjects and 114 control subjects ) were enrolled in the study . hypertension was diagnosed as a systolic blood pressure ( sbp ) 140 mmhg and/or a diastolic blood pressure ( dbp ) 90 mmhg , or the current use of antihypertensive medication . hypercholesterolemia was diagnosed as total cholesterol 5.2 mmol / l , and/or triglyceride 1.7 mmol / l and/or low density lipoprotein cholesterin 3.1 hba1c was measured on the same day that the samples were taken using a standard assay and a bio - rad variant hplc ii system ( bio - rad laboratories , hemel hempstead , uk ) , in accordance with the diabetes control and complication trial recommendations ( mitka , 2009 ) . all subjects were of han chinese origin from central china and belonged to independent pedigrees . informed consent was obtained from all participants and the study was approved by the institutional review board of xinxiang hospital . dna was extracted from peripheral blood by standard procedures and screened for the opg gene polymorphisms t950c , t245 g and g1181c using the polymerase chain reaction ( pcr ) and restriction fragment length polymorphism ( rflp ) , as previously described ( soufi et al . , 2004 ; pitocco et al . , 2009 ; biscetti et al . , 2012 ) . a 570-bp fragment around the t950c polymorphism was amplified with the primer pairs : 5-tgcgtccggatcttggctggatcgg-3 and 5-gggcgcggcgggcgcgcccagggacttaccacgagcgcgcagcacagcaa-3. the reaction was done in a final volume of 25 l that included 1 ng of genomic dna , 200 mol of dntp mixture / l , 0.2 mol of each primer / l , 2 gc buffer and 1 u of takara la taq dna polymerase ( takara biomedicals , dalian , liaoning , china ) . the pcr was done in a 2720 thermocycler ( applied biosystems , foster city , ca , usa ) with an initial denaturation at 95 c for 5 min , followed by 35 cycles of denaturation at 94 c for 30 s , annealing at 62 c for 30 s and extension at 72 c for 30 s , with a final extension at 72 c for 5 min . the pcr products were digested with hincii restriction endonuclease ( fermentas , burlington , on , canada ) at 37 c for 16 h and digestion products were separated on 2.5% agarose gels containing ethidium bromide . the 570 bp pcr product is cleaved into 288 bp and 282 bp fragments only in the presence of a c nucleotide at position 950 . the pcr primers for t245 g ( 271 bp fragment ) were 5-cgaaccctagagcaaagtgc-3 and 5-tgtctgattggccctaaagc-3. pcr was done as described above except that the annealing temperature was 56 c . the pcr products were digested with hinfi restriction endonuclease ( fermentas ) and separated on 3% agarose gels containing ethidium bromide . in the presence of a t nucleotide at position 245 , genotyping of the g1181c opg exon 1 polymorphism was done using a mis - matched oligonucleotide approach . a 570 bp fragment was amplified with the primers 5-tgcgtccggatcttggctggatcgg-3 and 5-gggcgcggcgggcgcgcccagggacttaccacgagcgcgcagcacagcta-3 , the latter containing a t instead of an a nucleotide two bases before the 3 end ; this position corresponds to the third base of codon 3 that encodes lysine in exon 1 of the opg gene and the substitution introduces an artificial xspi restriction site in the presence of the mutant allele . pcr was done as described above and the pcr products were digested with xspi restriction endonuclease ( fermentas ) for 16 h and subsequently separated on 3% agarose gels containing ethidium bromide . in the presence of a c nucleotide at position 1,181 , the 570 bp pcr product was cleaved into 522 bp and 48 bp fragments . to independently validate the pcr - rflp approach , we analyzed 49 randomly selected dna samples ( 10% of the total samples ) by direct sequencing and by pcr - rflp ; the results obtained with these two approaches were identical , indicating that the pcr - rflp method was reliable . chi - square tests and t - tests were used to compare proportions and means of baseline characteristics between cvd and control subjects . logistic regression was used to estimate the odd ratios ( ors ) and 95% cis for cvd risk , after adjusting for age , sex , body mass index ( bmi ) , hyper - cholesterolemia , smoking and hypertension . hardy - wein - berg equilibrium was assessed with the chi - square test or fisher s exact test , as appropriate . linkage disequilibrium was calculated using the software haploview 4.1 for all pairwise single nucleotide polymorphism ( snp ) combinations . all data analyses were done with spss 13.0 software ( spss inc . , chicago , il , usa ) for windows ( microsoft corp . , a value of p < 0.05 indicated significance and all statistical tests were two sided . all participants were randomly recruited from the department of internal medicine of the first affiliated hospital of xinxiang medical university in weihui ( henan province , people s republic of china ) , from september 12 , 2007 , to july 20 , 2012 . type 2 diabetes was diagnosed according to who criteria ( alberti and zimmet , 1998 ) . age at diagnosis of type 2 diabetes was > 40 years in 98% of the subjects and the minimum age at onset was 37 years . type 1 diabetes was carefully excluded on clinical grounds based on a review of medical records , on the fasting c - peptide levels and on the absence of islet - related auto - antibodies . for the purpose of this study , cvd was defined as the occurrence of a fatal or nonfatal myocardial infarction or coronary artery bypass grafting . subjects diagnosed with cvd before the diagnosis of type 2 diabetes were excluded , as were those diagnosed with stroke and/or angina . after these exclusions , 181 women ( 117 cvd case subjects and 64 control subjects ) and 309 men ( 195 cvd case subjects and 114 control subjects ) were enrolled in the study . hypertension was diagnosed as a systolic blood pressure ( sbp ) 140 mmhg and/or a diastolic blood pressure ( dbp ) 90 mmhg , or the current use of antihypertensive medication . hypercholesterolemia was diagnosed as total cholesterol 5.2 mmol / l , and/or triglyceride 1.7 mmol / l and/or low density lipoprotein cholesterin 3.1 hba1c was measured on the same day that the samples were taken using a standard assay and a bio - rad variant hplc ii system ( bio - rad laboratories , hemel hempstead , uk ) , in accordance with the diabetes control and complication trial recommendations ( mitka , 2009 ) . all subjects were of han chinese origin from central china and belonged to independent pedigrees . informed consent was obtained from all participants and the study was approved by the institutional review board of xinxiang hospital . dna was extracted from peripheral blood by standard procedures and screened for the opg gene polymorphisms t950c , t245 g and g1181c using the polymerase chain reaction ( pcr ) and restriction fragment length polymorphism ( rflp ) , as previously described ( soufi et al . , 2004 ; pitocco et al . , 2009 ; biscetti et al . , 2012 ) . a 570-bp fragment around the t950c polymorphism was amplified with the primer pairs : 5-tgcgtccggatcttggctggatcgg-3 and 5-gggcgcggcgggcgcgcccagggacttaccacgagcgcgcagcacagcaa-3. the reaction was done in a final volume of 25 l that included 1 ng of genomic dna , 200 mol of dntp mixture / l , 0.2 mol of each primer / l , 2 gc buffer and 1 u of takara la taq dna polymerase ( takara biomedicals , dalian , liaoning , china ) . the pcr was done in a 2720 thermocycler ( applied biosystems , foster city , ca , usa ) with an initial denaturation at 95 c for 5 min , followed by 35 cycles of denaturation at 94 c for 30 s , annealing at 62 c for 30 s and extension at 72 c for 30 s , with a final extension at 72 c for 5 min . the pcr products were digested with hincii restriction endonuclease ( fermentas , burlington , on , canada ) at 37 c for 16 h and digestion products were separated on 2.5% agarose gels containing ethidium bromide . the 570 bp pcr product is cleaved into 288 bp and 282 bp fragments only in the presence of a c nucleotide at position 950 . the pcr primers for t245 g ( 271 bp fragment ) were 5-cgaaccctagagcaaagtgc-3 and 5-tgtctgattggccctaaagc-3. pcr was done as described above except that the annealing temperature was 56 c . the pcr products were digested with hinfi restriction endonuclease ( fermentas ) and separated on 3% agarose gels containing ethidium bromide . in the presence of a t nucleotide at position 245 , genotyping of the g1181c opg exon 1 polymorphism was done using a mis - matched oligonucleotide approach . a 570 bp fragment was amplified with the primers 5-tgcgtccggatcttggctggatcgg-3 and 5-gggcgcggcgggcgcgcccagggacttaccacgagcgcgcagcacagcta-3 , the latter containing a t instead of an a nucleotide two bases before the 3 end ; this position corresponds to the third base of codon 3 that encodes lysine in exon 1 of the opg gene and the substitution introduces an artificial xspi restriction site in the presence of the mutant allele . pcr was done as described above and the pcr products were digested with xspi restriction endonuclease ( fermentas ) for 16 h and subsequently separated on 3% agarose gels containing ethidium bromide . in the presence of a c nucleotide at position 1,181 , the 570 bp pcr product was cleaved into 522 bp and 48 bp fragments . to independently validate the pcr - rflp approach , we analyzed 49 randomly selected dna samples ( 10% of the total samples ) by direct sequencing and by pcr - rflp ; the results obtained with these two approaches were identical , indicating that the pcr - rflp method was reliable . chi - square tests and t - tests were used to compare proportions and means of baseline characteristics between cvd and control subjects . logistic regression was used to estimate the odd ratios ( ors ) and 95% cis for cvd risk , after adjusting for age , sex , body mass index ( bmi ) , hyper - cholesterolemia , smoking and hypertension . hardy - wein - berg equilibrium was assessed with the chi - square test or fisher s exact test , as appropriate . linkage disequilibrium was calculated using the software haploview 4.1 for all pairwise single nucleotide polymorphism ( snp ) combinations . chicago , il , usa ) for windows ( microsoft corp . , redmond , wa , usa ) . a value of p < 0.05 indicated significance and all statistical tests were two sided . the demographic and clinical data for the type 2 diabetic subjects with and without cvd are shown in table 1 . univariate comparisons revealed no significant differences between the groups in terms of sex , age , bmi , hba1c , hypertension and hypercholesterolemia . in contrast , there were significantly more ( p = 0.02 ) smokers among diabetic patients with cvd than among those without cvd . table 2 shows the genotypic distribution of the t245c , t950c and g1181c gene polymorphisms . the genotype frequencies for all three polymorphisms were in hardy - weinberg equilibrium ( p > 0.05 ) in the cvd and control subjects . analysis with haploview 4.0 revealed no linkage disequilibrium for any pair - wise combination among the three selected snps . of the 178 patients with cvd , the genotype distributions of the t245 g and g1181c gene polymorphisms were not significantly different from those observed in the 312 subjects without cvd . similarly , the minor allelic frequency ( maf ) of the t245 g and g1181c gene polymorphisms were 0.11 and 0.34 in patients with cvd , which was not significantly different from that in subjects without cvd ( p = 0.88 and p = 0.73 , respectively ) . in contrast , there was a positive association between the t950c gene polymorphism and cvd in type 2 diabetic patients ( p = 0.04 for genotype and p = 0.02 for allele , respectively ) . based on this finding , we used unconditional logistic regression analysis to evaluate whether these gene variations were independent variables associated with cvd in type 2 diabetic patients . after adjusting for relevant confounding variables ( age , sex , bmi , hypercholesterolemia , smoking and hypertension ) , the c allele of the t950c polymorphism was found to be independently associated with the risk of cvd in type 2 diabetic patients in the additive model ( or = 1.38 , 95% ci 1.071.80 , p = 0.01 ) . however , there was no significant association for the t245 g and g1181c gene polymorphisms in the additive model ( or = 0.96 , 95% ci 0.641.45 , p = 0.79 and or = 1.06 , 95% ci 0.811.39 , p = 0.65 , respectively ) . after adjusting for relevant confounding variables , none of the three polymorphisms was associated with cvd in type 2 diabetic patients in the recessive or dominant model ( all p values > 0.05 ) . osteoprotegerin ( opg ) , a key factor in bone remodeling , is a member of the tumor necrosis factor ( tnf ) receptor family and a decoy receptor for the receptor activator of nuclear factor-b ligand ( rankl ) and tnf - related apoptosis inducing ligand ( trail ) . in addition to its role in the skeletal system , opg may have a role in vascular disease and has been implicated in human atherogenesis ( simonet et al . previous studies had found serum opg levels to be positively correlated with the presence and severity of cvd ( jono et al . , 2002 ; schoppet et al . , 2003 ) . clinical studies have shown that high serum opg levels are an independent risk factor for progressive atherosclerosis and cardiovascular diseases ( kiechl et al . , 2004 ; oh et al . , 2005 ) . for type 2 diabetic patients in particular , avignon et al ( 2005 ) reported an independent association between opg levels and asymptomatic coronary artery disease . opg gene polymorphisms have been associated with osteoporosis and vascular impairment ( hofbauer and schoppet , 2002 ) . furthermore , subjects with a c allele in the promoter region at position 950 ( tc and cc ) have significantly higher circulating opg serum levels , and genetic variations in the opg gene confer an increased risk of cvd and carotid plaque vulnerability in caucasians ( soufi et al . , ( 2006 ) observed that polymorphisms in the promoter region of the opg gene were not associated with aortic calcification or coronary artery disease in koreans . given this background , the aim of this study was to investigate the possible association between genetic variations in the opg gene and the risk of cvd in type 2 diabetic patients . this study is the first to show that the c allele of the t950c ( rs2073617 ) polymorphism in the opg gene is significantly and independently associated with an increased risk of cvd in type 2 diabetic patients . the t950c polymorphism is located 129 bp upstream from the tata box , 13 bp downstream from an activating protein 2 binding site and 32 bp upstream from a specificity protein 1 binding site . the biological significance of the association observed here is related to the fact that gene variants are functionally important . previous studies have shown that the c allele of the t950c ( rs2073617 ) polymorphism in the opg gene is significantly and independently associated with increased serum osteoprotegerin levels ( hofbauer and schoppet , 2002 ; straface et al . , 2011 ) . 2012 ) also showed that polymorphisms in the opg gene were associated with increased risk of ischemic stroke in diabetic patients . these findings further support that conclusion that osteoprotegerin is a risk factor for progressive atherosclerosis and cardiovascular disease not only in the general population but also in type 2 diabetic patients . this conclusion is consistent with the concept that the individual chances of presenting an atherosclerotic - related disease may be affected by a susceptibility profile that is genetically defined ( brito et al . , 2009 ; ding and kullo , 2009 ; mollsten et al . , 2009 ) . these data further suggest a role for opg as a reliable biomarker in cardiac and vascular disease . although the mechanisms linking opg and vascular disease require further study , the association between opg and cvd in type 2 diabetic patients shown here requires further investigation to clarify the possible role of opg as a biomarker for identifying patients with , or at risk of , cardiovascular events . first , the sample size in this study was relatively small and could yield a false positive result . in addition , the relatively small sample size and rare frequency of the t245 g polymorphism may have resulted in insufficient statistical power to detect a positive association . second , this was a case - control study . consequently , recruitment and survival bias can not be excluded , particularly in the control population in which the possibility that some of the controls might develop cardiovascular disease in the future could not be eliminated . third , this study was restricted to han chinese in order to avoid the possible confounding effect of race . for other ethnic groups or races , other genetic markers the choice of appropriate markers requires knowledge of the differences in linkage disequilibrium patterns among races . in summary , osteoprotegerin gene polymorphisms associated with cvd in the general population were also associated with cvd in diabetic subjects . although diabetic and non - diabetic individuals showed similarities in their genetic susceptibility to cvd , diabetic patients had certain peculiarities in their genetic architecture that influenced their susceptibility to cvd .
osteoprotegerin ( opg ) gene polymorphisms ( t245 g , t950c and g1181c ) have been associated with osteoporosis and early predictors of cardiovascular disease . the aim of this study was to evaluate whether these polymorphisms contribute to cardiovascular disease ( cvd ) in type 2 diabetic patients . we performed a case - control study with 178 cvd subjects with diabetes and 312 diabetic patients without cvd to assess the impact of variants of the opg gene on the risk of cvd . the opg gene polymorphisms were analyzed by using the polymerase chain reaction ( pcr ) and restriction fragment length polymorphism ( rflp ) . there was no significant association between the t245 g and g1181c polymorphisms and cvd in the additive genetic model ( or = 0.96 , 95% ci 0.641.45 , p = 0.79 ; or = 1.06 , 95% ci 0.811.39 , p = 0.65 , respectively ) . however , the c allele of the t950c polymorphism was independently associated with a risk of cvd in type 2 diabetic patients in this genetic model ( or = 1.38 , 95% ci 1.071.80 , p = 0.01 ) . this study provides evidence that the c allele of the t950c polymorphism is associated with increased risk of cvd in diabetic patients . however , well - designed prospective studies with a larger sample size are needed to validate these results .
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Proceed to summarize the following text: hydrogen sulfide is a recently identified gasotransmitter reported to have numerous physiological effects in diverse processes including metabolism , inflammation , the nervous system , and the cardiovascular system . the cardiovascular effects of this molecule are currently of major interest and include vascular relaxation , cardioprotection , and vasculoprotective effects [ 2 , 3 ] . in mammalian cells , h2s is produced primarily by 2 pyridoxyl-5-phosphate - dependent enzymes , cystathionine--synthase ( cbs ) , and cystathionine--lyase ( cse ) . additionally , a role for 3-mercaptopyruvate sulfurtransferase in concert with cysteine aminotransferase has been identified in the vasculature . with respect to vasoregulation , cse is of particular interest as it is reported to be present in a range of vascular beds and its expression has been clearly identified in vascular smooth muscle cells . cse has also been located in endothelial cells and additionally it is reported to contribute to endothelium - dependent vasorelaxation [ 5 , 6 ] . inhibition of cse with the irreversible inhibitor d , l - propargylglycine ( ppg ) leads to an elevation of blood pressure in vivo and increased vascular tone in vitro . importantly , mice deficient in cse are hypertensive compared to their wild - type controls . collectively , these reports point to a role for h2s in the regulation of vascular function . atherosclerosis is the most common underlying cause in the development of coronary artery disease , a leading cause of death and morbidity worldwide . atherosclerosis is a chronic immunoinflammatory , fibroproliferative disease caused by lipid accumulation , affecting large- and medium - sized arteries [ 9 , 10 ] . atherosclerosis has a multifactorial pathogenesis , involving vascular inflammation , including increased reactive oxygen species generation which leads to impairment of vascular endothelial function , by reducing no bioavailability . a number of previous studies have indicated that h2s has many properties that may lead to the inhibition of atherogenesis ( for review see ) . these properties include inhibition of proliferation , induction of apoptosis in vascular cells , inhibition of oxidative damage , and decreased production of reactive oxygen species ( ros ) [ 15 , 16 ] . these effects are all cytoprotective , and additionally h2s treatment leads to decreased foam cell production , a reduction in adhesion molecule expression decreased chemokine signalling and decreased platelet aggregation . h2s is also reported to have antiinflammatory and anti - remodelling effects , in addition to vasorelaxant effects in both large [ 8 , 2325 ] and small [ 2628 ] blood vessels which could also be beneficial in preventing the progression of vascular disease . this study focuses on the anti - oxidant properties of h2s that suggest that it may be a useful agent in the treatment or prevention of vascular dysfunction in atherosclerosis . thus , our hypothesis was that h2s will act as a vascular protective factor , due to inhibitory effects on superoxide production and action in the vasculature . the specific aims of this study were to examine the role of both endogenously produced h2s and chronic exogenous h2s treatment in vivo on a lesion development , vascular superoxide generation , and endothelial function in the fat - fed apoe mouse model of atherosclerosis . all experimental procedures involving the use of animals were carried out in accordance with the guide for the care and use of laboratory animals published by the us national institutes of health and the project was approved by the rmit university animal ethics committee prior to the commencement . all animals were housed in the rmit animal facility , rmit university , bundoora west campus , on a 12-hour day / night cycle at room temperature of 20 2c . male apoe mice with a c57bl/6j background ( n = 64 ) and male c57bl/6j wild - type ( wt ) mice were purchased from the animal resource centre , western australia . from 5 weeks of age mice were fed a high - fat diet containing 22% fat , 0.15% cholesterol ( specialty feeds , western australia ) for 16 weeks . during the final four weeks of fat feeding , mice were assigned to 4 groups : ( 1 ) untreated , ( 2 ) treated with nahs to deliver h2s at 10 mol / kg / day , ( low nahs ) , ( 3 ) nahs 100 mol / kg / day ( high nahs ) , or ( 4 ) d , l - propargylglycine ( ppg ) 30 mg / kg / day . adult male c57 mice ( n = 6 ) , aged 16 weeks and fed on standard rodent chow , were also used as a wild - type ( wt ) control group . systolic blood pressure was measured at weeks 12 and 16 ( before and after treatment ) , using the noninvasive tail - cuff apparatus ( adinstruments , sydney ) . systolic blood pressure was averaged from four to six consecutive measurements taken at intervals of 1 - 2 mins . mice were culled in a humane manner via co2 asphyxiation ( 95% co2 , 5% o2 ) , followed by cervical dislocation and decapitation . the aorta and liver were dissected out and washed in ice - cold oxygenated krebs ' solution ( composition in mm : nacl 119 , kcl 4.7 , mgso4 1.17 , nahco3 25 , kh2po4 1.18 , cacl2 2.5 , glucose 5.5 , edta 0.026 , and ph 7.4 ) . aortic arches ( for oil red o staining ) and abdominal aorta segments ( for endothelial function assays ) were used immediately . total plasma cholesterol , high - density lipoprotein ( hdl ) , low - density lipoprotein ( ldl ) , and triglyceride levels were measured with a konelab 20xti random access analyser . development of atherosclerotic lesions across the aortae was assessed using en face staining with oil red o. the aortae were dissected free of connective tissue and stained with oil red o then washed in 60% isopropyl alcohol . image j was used for analysis of total tissue to lesion area ratio via manual tracing with measurements based on a calibration image of 1 mm . abdominal aortic rings were mounted in myograph chambers , where they were maintained in krebs ' solution at 37c , continuously supplied with carbogen ( 95% co2 , 5% o2 ) . the aortic rings were allowed to equilibrate for 20 min under zero force , then a 5 mm resting tension was applied . changes in isotonic tone were recorded using myograph interface model 610 m ( adinstruments , sydney ) and the myodac data acquisition system . concentration response curves to the endothelium - dependent dilator acetylcholine ( ach ) were constructed in vessels preconstricted with the thromboxane analogue u46619 . precontraction to u46619 was submaximal and not significantly different between groups . at the end of the ach curve , 10 m of the endothelium - independent dilator levcromakalim ( lkm ) was added to test vascular smooth muscle cell function . superoxide anion production in the aorta was determined by lucigenin - enhanced chemiluminescence assay as previously described [ 29 , 30 ] . briefly , sections of abdominal aorta ( 3 mm long ) were preincubated for 45 min at 37c in krebs - hepes buffer ( composition ( mm ) : nacl 99.9 , kcl 4.7 , kh2po4 1.0 , mgso47h2o 1.2 , d - glucose 11.0 , nahco3 25.0 , cacl22h2o 2.5 , na hepes 20.0 , and ph 7.4 ) containing diethyldithiocarbamic acid ( 1 mm ) to inactivate superoxide dismutase and nadph ( 100 m ) as a substrate for nadph oxidase . 300 l of krebs - hepes buffer , containing lucigenin ( 5 m ) with and without the flavoprotein inhibitor diphenylene iodonium ( dpi , 1 m ) to inhibit nadph oxidase , was placed into a 96-well optiplate , which was loaded into a polarstar optima photon counter ( bmg labtech , melbourne , vic , australia ) to measure background photon emission at 37c . after background counting was completed , a single ring of aorta was added to each well and photon emission was recounted . the background reading was subtracted from the superoxide anion counts and normalized with dry tissue weight . results are expressed as mean standard error of the mean ( sem ) with the number of experiments denoted by n. concentration response curves to ach were expressed as a percentage reversal of the u46619 precontraction . these data were computer fitted to a sigmoidal curve using nonlinear regression ( graphpad prism , version 5.0 ) to calculate the sensitivity of the vasorelaxation response ( pec50 ) . statistical analysis was performed using either unpaired t - tests or by 1-way analysis of variance ( anova ) with post hoc tests applied as appropriate and as stated in the text ( graphpad prism , version 5 , graphpad software incorporated ) . all drugs were dissolved in dh2o , with the exception of lkm , which was dissolved in methanol . systolic blood pressure measurements were recorded from fat - fed apoe mice in each treatment group at weeks 12 and 16 ( before and after treatment ) and in age - matched wt controls at 16 weeks . fat - fed apoe mice had significantly higher systolic blood pressure than the age - matched wt controls . fat - fed apoe mice treated with either 10 or 100 mol / kg / day nahs had a significant reduction in systolic pressure compared to untreated mice ( figure 1 ) . plasma lipid profiles were collected from fat - fed apoe mice in each treatment group and also from an age matched wt control group . high - fat - fed apoe mice had significantly greater total cholesterol , and ldl levels when compared to wt controls ( table 1 ) . treatment with either 10 or 100 mol / kg / day nahs had no effect on plasma lipid profile nor did treatment with ppg ( 30 mg / kg / day ) . the luminal surfaces of the aortic arches from the high - fat - fed apoe mice in each treatment group were examined for total atherosclerotic lesion area . mice treated with nahs ( 10 mol / kg / day ) showed a significant 8% decrease in lesion area when compared with the untreated high - fat - fed apoe mice . treatment with ppg had no significant effect on total lesion area ( figure 2 ) . isolated abdominal aorta from high - fat - fed apoe mice displayed marked endothelial dysfunction compared to wt mice as sensitivity and maximum vasorelaxation responses ( rmax ) to the endothelium - dependent dilator ach were significantly impaired ( table 2 , figure 3 ) . chronic treatment with either dose of nahs significantly improved endothelial function compared to untreated fat - fed apoe mice , as both rmax and pec50 values for these groups were significantly improved and comparable to wt controls . the group treated with ppg had a suppressed rmax and also displayed impairment in sensitivity to ach which was similar to that in untreated fat - fed apoe . relaxation responses to the no donor sodium nitroprusside ( snp 10 m ) were maximal and of equivalent sensitivity in all treatment groups ( data not shown ) . aortic rings from wt and apoe mice from each treatment group were collected for determination of vascular superoxide generation . fat - fed apoe mice had significantly greater vascular superoxide production than age - matched controls . there was a significant inhibition of superoxide production in the 10 and 100 mol / kg / day nahs - treated groups ( figure 4 ) . the primary finding of this study is that chronic nahs treatment in vivo has a vasoprotective effect in mice under conditions of high - fat diet and genetic dyslipidaemia . this is the first study to show that chronic exogenous nahs treatment in vivo can cause a reduction in vascular superoxide anion generation , which has the effect of protecting the endothelium from damage thereby preserving endothelial function . in addition , these data show the benefits of nahs treatment also extend to inhibiting the development of vascular lesions and reducing systolic blood pressure . hydrogen sulfide has emerged as an important cardiovascular mediator that has been shown to modulate vascular tone and blood pressure . deficiency of endogenous h2s is reported to play a role in the development of hypertension in spontaneously hypertensive rats and cse - deficient mice are reported to be hypertensive . the reduction in systolic blood pressure seen in the present study is modest , but significant , and likely results from the vasorelaxant effects of nahs . these have been attributed primarily to actions at katp channels , but other k channel subtypes [ 8 , 23 , 26 , 32 ] , ca channels and other mechanisms involving pde and pkg are also implicated . a most important finding from this study endothelium dependent vasorelaxation is significantly impaired in apoe mice fed a high - fat diet , with both sensitivity and maximal relaxation significantly attenuated . indeed , endothelial dysfunction is a feature of early blood vessel disease and occurs before the development of atherosclerotic lesions . chronic treatment with nahs restores endothelial function to the same level as in the wild - type mice . this probably indicates a protection of no bioavailability since acetylcholine initiated endothelium - dependent vasorelaxation is primarily mediated by no in mouse aorta and interestingly , the protective effects of nahs on endothelial function occur at the same dose as the inhibition of vascular superoxide production , suggesting a link between these two events , and consistent with the fact that increased superoxide is known to inhibit no bioavailability . h2s is a potent one - electron chemical reductant and nucleophile that is theoretically capable of scavenging free radicals by single electron or hydrogen atom transfer . thus , h2s may participate in many reactions and is reported to readily scavenge reactive oxygen and nitrogen species such as peroxynitrite , superoxide , hydrogen peroxide , hypochlorous acid and lipid hydroperoxides . additionally it has been demonstrated that h2s can inhibit the activity and expression of nadph - oxidase , the major vascular source of superoxide . it is well known that reactive oxygen species contribute to the pathogenesis of cardiovascular diseases . the parent reactive oxygen species is the free radical superoxide produced by several oxidases including nadph oxidase , xanthine oxidase , cyclooxygenase and endothelial nitric oxide synthase ( enos ) in its uncoupled state . overproduction of reactive oxygen species , in particular superoxide from nadph oxidase , is implicated as a key mediator of endothelial dysfunction and loss of no bioavailability is associated with many cardiovascular diseases , including diabetic vascular disease , hypertension and atherosclerosis . that oxidative stress apparent in atherosclerosis was affirmed in this study as the fat - fed apoe mice had significantly greater vascular superoxide generating capacity than the wild - type controls . the lucigenin - based assay employed here showed that the presence of the flavoprotein inhibitor dpi , used to inhibit nadph oxidase , almost abolished the vascular superoxide production , strongly suggesting that the source of superoxide was an nadph oxidase . previous work has shown that nahs inhibits activity and expression of nadph - oxidase [ 16 , 46 ] in cultured vascular smooth muscle cells however the present study extends this finding suggesting an in vivo effect of nadph oxidase inhibition by chronic nahs treatment and offers a likely molecular mechanism for the vasculoprotective effect of chronic nahs treatment in this study . this is not surprising as the fat feeding regime used provides a gross excess of dietary cholesterol . in this model of atherosclerosis , advanced lesions covering nearly 40% of the lumen of the aorta were observed in mice fat - fed for 16 weeks . these vascular lesions were substantially reduced with nahs treatment for the final 4 weeks of fat feeding , suggesting that the treatment may not just inhibit the progression of lesion development but may also cause regression of lesions . this effect is possibly related to the inhibition of inflammatory responses involving nf-b and adhesion molecule expression . additionally , this effect may be due to suppression of vascular superoxide production , since previous studies in this model show a link between atheroprotection and reduced vascular superoxide with consequent increased no bioavailability [ 4850 ] . d , l - propargylglycine is a widely used inhibitor of cse , despite quite poor selectivity and cell permeability . the dose of ppg used in this study is the same as that used previously in apoe mice and is maximal as further increases in dose can elicit nephrotoxicity . in the current study cse activity in the liver , the richest tissue source of the enzyme , was shown to be markedly suppressed in the ppg - treated group ( data not shown ) , suggesting that vascular cse would also be suppressed , although we did not test this directly . on the basis of using ppg as an inhibitor of cse , endogenous h2s is reported to be involved in the regulation of basal blood vessel tone and indeed blood pressure . previous studies examining the progression of atherosclerosis in apoe mice found that cse activity and endogenous h2s production were inhibited in this model , results that concur with other data showing an inverse relationship between plasma h2s levels and cardiovascular disease . found that treatment with ppg exacerbated the atherogenesis in their apoe mouse model . ppg did not have any exacerbating effect , suggesting that cse - derived h2s does not ameliorate the deleterious effects of the combination of the atherogenic diet and genetic dyslipidemia on blood pressure or vascular dysfunction in this model of atherosclerosis . a simple explanation for this is that endogenous h2s would be overwhelmed by the atherogenic insult of the model , which adds a high - fat diet to the genetic dyslipidaemia , a significant enhancement of the atherogenic potency . it is also possible that other non - cse sources of h2s , are upregulated under conditions of cse inhibition , to replace cse - derived h2s these may include h2s derived from cystathionine - beta synthase or 3-mercaptopyruvate transferase and further studies would be required to investigate this possibility . more work is still required to examine the pharmacokinetics of nahs and the distribution , sequestration , and metabolism of h2s donated from any of the reported h2s donor compounds . it is noted that the present results are obtained from a single daily ip dose of nahs for a relatively short period . nahs rapidly forms h2s when dissolved , and it has been shown that intravenously administered nahs is rapidly removed from the plasma . h2s is rapidly consumed in oxygenated tissues , so it was not expected that plasma concentrations of sulfide would be different between groups . plasma h2s levels were not measured in this study since we reasoned that they were unlikely to be different between groups and there is controversy over the methods available for doing this . despite this , previous studies indicate that treatment with similar doses of nahs in apoe mice does indeed increase plasma h2s levels . that aside , in this study the administered nahs certainly causes an acute effect which then has longer - term consequences . there is evidence that sulfide donors can cause sulfhydration of specific proteins , and though speculative , this is a potential mechanism that would be the starting point for further investigation . in the present study a dose dependence of nahs effect is not apparent for any of the parameters investigated . the beneficial effects of nahs in reducing blood pressure , improved endothelial function , decreased lesion area and vascular superoxide production were all observed at a dose of 10 mol / kg / day and this effect was preserved but not enhanced at 100 mol / kg / day for the sbp data , endothelial function data , and reduced vascular superoxide production , suggesting that the effects on blood vessel tone , endothelial protection , and vascular superoxide production may be linked . these data also indicate that there is a tight therapeutic window for the effects of a sulfide donor in this condition , an observation that is supported by several previous studies ( see ) . the field of h2s biology is plagued with a lack of tools , in particular selective and specific blockers of h2s - producing enzymes , consistent and stable donors and reliable scavengers . improvements in these will be most useful for advances in the study of the physiological and pathophysiological effects of h2s . in conclusion , this is the first study to show in an in vivo model an inhibition of vascular superoxide generation with chronic nahs treatment . this effectively protects endothelial function from the oxidative stress induced by the atherogenic diet and renders the vessels resistant to the development of atherosclerotic lesions . an additional beneficial effect of chronic nahs treatment is a reduction in systolic blood pressure , probably due to vasorelaxant effects . in conclusion , these data show that nahs donors may be a useful prevention and treatment for vessel damage caused by oxidative stress .
hydrogen sulfide is emerging as an important mediator of vascular function that has antioxidant and cytoprotective effects . the aim of this study was to investigate the role of endogenous h2s and the effect of chronic exogenous h2s treatment on vascular function during the progression of atherosclerotic disease . apoe/ mice were fed a high - fat diet for 16 weeks and treated with the h2s donor nahs or the cystathionine--lyase ( cse ) inhibitor d , l - propargylglycine ( ppg ) , to inhibit endogenous h2s production for the final 4 weeks . fat - fed apoe/ mice displayed significant aortic atherosclerotic lesions and significantly impaired endothelial function compared to wild - type mice . importantly , 4 weeks of nahs treatment significantly reduced vascular dysfunction and inhibited vascular superoxide generation . nahs treatment significantly reduced the area of aortic atherosclerotic lesions and attenuated systolic blood pressure . interestingly , inhibiting endogenous , cse - dependent h2s production with ppg did not exacerbate the deleterious vascular changes seen in the untreated fat - fed apoe/ mice . the results indicate nahs can improve vascular function by reducing vascular superoxide generation and impairing atherosclerotic lesion development . endogenous h2s production via cse is insufficient to counter the atherogenic effects seen in this model ; however exogenous h2s treatment has a significant vasoprotective effect .
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Proceed to summarize the following text: the centers for disease control and prevention ( cdc ) has a mandate to be prepared to analyze large numbers of samples in the event of a poisoning of the population by a wide range of chemical threat agents including nerve agents . one of the current cdc methods to assess exposure to nerve agents uses mass spectrometry to analyze adducts on plasma butyrylcholinesterase ( bche ) . bche is a sensitive biomarker of exposure , capable of scavenging subclinical doses of nerve agents and other organophosphorus toxicants ( ops ) . a substantial proportion of the american and european population ( 35% ) carries at least one mutated bche allele . one goal of this review is to evaluate difficulties that might arise for analysis of exposure when the plasma contains genetic variants of bche . acute toxicity from exposure to cholinesterase inhibitors is attributed to the inhibition of acetylcholinesterase ( ache ) activity at the neuromuscular junction and in the brain , resulting in depression of the respiratory and circulatory centers in the medulla , weakness of the muscles of respiration , and pulmonary edema . since toxicity correlates with depression of ache activity rather than bche activity , it would seem logical to focus on genetic variants of ache . though genetic variants of human ache exist , deleterious mutations are rare and occur only in the heterozygous state . furthermore , ache is not readily accessible for study because ache is membrane bound to red blood cells ( rbc ) . a second goal of this review is to assess the effect of genetic variants of bche on susceptibility to cholinesterase inhibitors . we examine the evidence for the common assumption that humans with bche deficiency are at increased risk of toxicity from op exposure . hundreds of op poisoning cases occur annually in sri lanka , india , and china , but their bche genotype is unknown . a single study did measure the bche genotype in brazilian farmers and correlated the bche genotype with ache activity levels in rbc as an indicator of pesticide exposure . as of the year 2016 , there is no conclusive evidence that individuals with bche deficiency are at increased risk of toxicity from op pesticides . gene sequences for 60,706 unrelated individuals are available online from the exome aggregation consortium ( exac ) http://exac.broadinstitute.org/gene/ensg00000114200 . the database shows mutations in exons 2 , 3 , 4 , 5 , and 6 for ache but does not include upstream , untranslated exons . a summary of the number of mutations in human ache and bche exons can be found in table 1 . pli is the probability of a gene being tolerant to loss - of - function . a pli score of 1.0 indicates intolerance of mutations that result in loss of function . a pli score of 0.0 indicates tolerance of mutations that result in loss of function . the most abundant missense mutation , his353asn ( h322n ) , has an allele count of 4,910 out of 115,962 for a frequency of 0.04234 . ( the residue number in parentheses is for the secreted protein from which the signal peptide has been deleted . ) this mutation defines the yt blood group antigen but has no effect on ache activity . the residues involved in activity and substrate interactions for ache have been well characterized . missense mutations are absent from residues known to be important for ache enzyme activity , with minimum exceptions . the catalytic triad residues , ser234 ( s203 ) , glu365 ( e334 ) , and his478 ( h447 ) are conserved in the exac study with the exception of one his478 allele out of 111,128 , mutated to his478gln . the peripheral site residues trp317 ( w286 ) , asp105 ( d74 ) , tyr372 ( y341 ) , and phe328 ( f297 ) are conserved . residues that constitute the oxyanion hole , gly151 ( g120 ) , gly153 ( g122 ) , and ala235 ( a204 ) , are conserved with the exception of 2 alleles out of 116,198 for ala235 that are mutated to ala235thr . the missense mutation asp165his ( d134h ) destabilizes the ache enzyme at temperatures higher than 31 c . this variant has normal catalytic activity with acetylthiocholine but is inhibited 2-times more slowly by paraoxon compared to wild - type ache and is reactivated 4-times faster by the oxime 2-pam . the frequency of the asp165his mutant is so low that it was not found in the consortium study of 60,706 individuals . the remarkable finding for the ache gene is that it is almost devoid of loss of function mutations . the exac database identified 2 splice acceptor mutations and one frameshift mutation in the ache gene . only one allele in the set has a frameshift mutation , and that mutation is in the heterozygous state . exons 2 , 3 , 4 , and 5 code for the glycolipid - anchored form of ache bound to the red blood cell membrane . the ache that functions in nerve impulse transmission is encoded by exons 2 , 3 , 4 , and 6 and completely bypasses exon 5 . the absence of loss of function mutations in the human ache gene spliced to exon 6 , isoform e4-e6 , is evidence for extreme selective constraint . if this interpretation is correct , it is puzzling why ache knockout mice , with no ache activity in any tissue , are born alive and live to adulthood , though they are weak , blind , suffer convulsions , and do not reproduce . additionally , no disease has been attributed to a mutation in the human ache enzyme . mutation data for the 60,706 unrelated individuals in the exac report are summarized in table 1 . in contrast to ache , the bche gene was observed to have 34 loss of function mutations , though none was in the homozygous state in the exac database . bche was assigned a pli score of 0.0 , indicating tolerance to loss of function mutations in the bche gene . this conclusion is supported by the many reports of silent bche in humans , where the individual has no bche activity due to frameshift , splice junction , stop , or missense mutations . humans homozygous for silent bche are healthy , fertile , and live to old age . five alleles out of 121,110 had ser226gly ( s198 g ) in the active site serine , a mutation that results in complete loss of bche activity . one allele out of 121,174 had a mutation in his of the catalytic triad : his466arg ( h438r ) . one bche allele out of 121,142 had the mutation trp110arg ( w82r ) at the choline binding site , a mutation expected to severely impair bche activity . the missense mutation asp98gly ( d70 g ) is responsible for prolonged apnea in response to the muscle relaxants succinylcholine and mivacurium . the mutation is cataloged as rs1799807 in the national center for biotechnology information database . this is the atypical variant recognized by kalow as having poor affinity for succinylcholine . individuals who are homozygous for atypical bche are unable to breathe for 2 h from a dose of succinylcholine that paralyzes most people for 3 min . other , less frequent mutations including those that produce silent bche with no activity are also associated with prolonged apnea in response to a muscle relaxant . these additional mutations and the case studies that report them have been recently reviewed . a recent case study of six patients described new bche mutations associated with prolonged apnea following administration of mivacurium . the most frequent missense mutation , ala567thr ( a539 t ) the k - variant named in honor of werner kalow , is located in the tetramerization domain near the c - terminus of bche . the recombinant k - variant has wild - type bche activity and stability , leading to the hypothesis that the reduced plasma activity is explained by an unknown mutation in a regulatory region rather than by the ala567thr mutation . the netherlands organisation for applied scientific research ( tno ) prins maurits laboratory and the cdc have both developed sensitive mass spectrometry methods for detecting nerve agent and op pesticide exposure by measuring adducts on the active site serine of bche . human ache and bche have the same fgesagaas sequence . naturally occurring mutations in fgesagaas alter the mass of the parent ion and therefore would be missed in mass spectrometry methods set up to search for parent ion masses specific for the wild - type active site peptide . table 2 lists naturally occurring missense mutations in the fgesagaas peptide in human ache and bche . the frequency of ache mutations in the fgesagaas peptide is 2 in 116,000 alleles . the frequency of bche mutations is slightly higher at 9 out of 121,000 alleles and includes mutations that result in the complete absence of bche activity . the 60,706 individuals in the exac study population had no homozygous silent bche , though inbred groups in alaska and india include a high proportion of individuals who are completely deficient in bche activity . in a population of about 5,000 eskimos in western alaska , 39 persons completely lacked bche activity , and 24 persons had very low ( 210% of normal ) bche activity . a study of 490 persons belonging to the vysya caste of andhra pradesh , india found 12 homozygous silent persons and 85 heterozygotes for a frequency of 0.1112 for the silent bche allele . ache levels are highest in the brain , followed by skeletal muscle ( figure 1a ) , consistent with the function of ache in nerve impulse transmission . bche levels are highest in the liver , adipose - visceral , esophagus , colon , fallopian tube , uterus , cervix , and lung ( figure 1b ) , consistent with the function of bche in detoxication of poisons that are eaten or inhaled . whole body autoradiograms of mice injected intravenously with h - soman showed that 5 min after administration , a high concentration of radioactivity was in the blood , heart , kidney , lung , nasal cavities , lacrymal glands , salivary glands , skin , and some striated muscles . when enzyme activity levels are multiplied by the mass of the tissue , the highest quantity of ache enzyme is in muscle , whereas the highest levels of bche are in the plasma and liver . overall , the adult human body has 10-times more bche protein ( 680 nmol ) than ache protein ( 62 nmol ) . nerve agents and op pesticides are structurally similar in that they are esters of phosphoric acid . op pesticides that have a p = s bond ( for example , parathion ) are inactive as cholinesterase inhibitors until they are metabolically converted to the oxon ( paraoxon ) . exposure to nerve agents has immediate toxic consequences , whereas exposure to op pesticides has delayed toxic consequences reflecting the time it takes the cytochrome p450 enzymes to replace the p = s bond with p = o . nerve agents are selected for their toxicity to mammals , whereas op pesticides are selected for their toxicity to pests . at the clinical level , the early symptoms of nerve agent and op pesticide intoxication are identical , and treatment strategies for both types of poisoning are similar . the acute toxicity from nerve agents and op pesticides is due to the inhibition of ache activity , resulting in the accumulation of acetylcholine . nerve agents and op pesticides make a covalent bond with the active site serine of ache and bche . the added mass from the reaction with nerve agents is different from the added mass from the reaction with op pesticides , making it possible to distinguish between these classes of poisons using mass spectrometry . a main difference between nerve agents and op pesticides is the dose that inhibits ache . very low doses of nerve agents are inhibitory , whereas only high doses of op pesticides are inhibitory . thus , nerve agents are several orders of magnitude more acutely toxic than most op pesticides . a , title x , section 1078 passed by the 105th us congress in november 1997 , restricted the use of human subjects for testing of chemical agents . hundreds of young men had been tested with low doses of nerve agents in the years 1948 to 1967 , though approval to publish findings was delayed about a decade . many of the reports have never been published , but their contents are summarized in a chapter by frederick sidell . most studies quantified nerve agent exposure by measuring the depression of ache activity in rbc , a surrogate for ache activity in the neuromuscular junction and brain . toxic symptoms were monitored , and it was found that the same low dose of nerve agent affected individuals differently . one of the earliest studies in the open literature reported the effects of the nerve agent sarin administered orally to 10 volunteers . the study established that ache in rbc and bche activity in plasma are irreversibly inhibited by nanomolar concentrations of sarin . toxic symptoms correlated with the level of inhibition when the subject was first exposed , but ache activity in rbc could be gradually depressed to near zero by repeated doses administered over a period of several days without symptoms necessarily ensuing . symptoms usually disappeared before any restoration of rbc ache or plasma bche activity . plasma bche activity returned to normal in 40 days , but ache activity in rbc took 92 days to return to its original value . a study conducted in 1965 and approved for publication in 1974 measured the effects of small doses of the nerve agents vx and sarin administered to us army enlisted men , 2028 years old , who volunteered with no compensation thirty - four subjects were given vx intravenously , 32 subjects drank vx , and 23 subjects received sarin intravenously . for example , of the 18 subjects who were given 1.5 g / kg sarin intravenously , 11 had mild , transient symptoms of dizziness beginning within 12 min of injection and lasting 1015 min . rbc ache activity ranged from 9 to 33% of normal for the latter 6 subjects and 1444% for the asymptomatic subjects , suggesting insignificant correlation between rbc ache inhibition and toxic symptoms . it was found that vx was 3-times more potent than sarin ; the dose that inhibited 50% of the rbc ache was 1.1 g / kg vx administered intravenously and 3 g / kg sarin . rbc ache activity spontaneously reactivated from inhibition by vx , and its reactivation rate was assisted by the administration of 2-pyridinium aldoxime methochloride ( 2-pam ) with a recovery of 70% of the initial ache activity . an accidental exposure case affected one man who inhaled a low dose of vx vapor in a military laboratory . the subject exhibited early clinical signs of nerve agent intoxication including tightness in the chest , difficulty breathing , blurred vision , mild rhinorrhea , and eyelid muscle fasciculations . all symptoms disappeared within 24 h. activity assays showed no depression of rbc ache activity . however , the rbc fraction of whole blood collected on day 1 yielded 219.9 pg / ml of the fluoride analogue of vx , while the plasma fraction yielded 81.2 pg / ml . samples taken as late as 27 days following exposure yielded detectable quantities of the vx analogue in the rbc fraction as well as in plasma . in this case , the fluoride reactivation method coupled to analysis by gas chromatography mass spectrometry was clearly a more sensitive method for detecting vx exposure than ache and bche activity assays . ache and bche activity assays , though less sensitive indicators of exposure than mass spectrometry methods , are run because the enzyme activity assays are simple , quick , and relate to decades of literature . the aum shinrikyo release of sarin in the tokyo subway in 1995 poisoned 5,000 people and killed 12 . within 2 h of the event , doctors had correctly diagnosed nerve agent exposure . severely ill patients had reduced plasma bche activity and the classic symptoms of nerve agent exposure . reduced rbc ache activity was noted in 34 out of 66 patients . the speed with which physicians established the cause of illness saved lives because they knew to treat nerve agent intoxication with atropine and 2-pam . in their publications , the japanese researchers involved with the event acknowledged the usefulness of previously published reports describing the effects of sarin in man and treatment of nerve gas poisoning . human studies with known doses of sarin had used only low doses that caused mild to moderate symptoms . some of the victims in the tokyo subway inhaled doses that resulted in coma and convulsions . the incident provided new information on survival from high doses of sarin and the consequent impairment of learning , memory , and personality change . following this event , scientists developed new methods to detect nerve agent exposure based on analysis of adducts on bche , release of adducts with potassium fluoride , and analysis of metabolites in urine and serum . bche with the single amino acid substitution asp98gly ( d70 g ) is henceforth called atypical bche to conform with 60 years of scientific literature . individuals homozygous for atypical bche have prolonged apnea of 2 h after a standard dose of succinylcholine or mivacurium . substrates and inhibitors that carry a positive charge have a poor affinity for atypical bche . the positively charged nerve agent vx inhibits atypical bche 21-fold more slowly than vx inhibits wild - type bche . atypical bche is resistant to inhibition by the positively charged carbamate poison from the calabar bean , physostigmine , and the positively charged drugs neostigmine and physostigmine . inhibition of 50% of the atypical bche activity in human serum requires 20-fold higher concentrations of physostigmine and neostigmine than for wild - type bche . rate constants for the inactivation of atypical bche by physostigmine and pyridostigmine are 14- and 7-fold slower than that for wild - type bche . the muscle relaxant succinylcholine carries two positive charges ; its ic50 is 100-fold higher for atypical bche compared to that of wild - type bche . inhibitors that carry no charge are equally effective inhibitors of wild - type and atypical bche . for example , the nerve agents sarin and soman inhibit wild - type and atypical bche with the same rate constants . similarly , paraoxon , the active metabolite of the pesticide parathion , makes no distinction between wild - type and atypical bche in rate of inhibition . organophosphorus and carbamate inhibitors make a covalent bond with the active site serine of bche . carbamate - inhibited bche spontaneously regains full activity with a half - life of about 23 h. organophosphorus nerve agents , having lost the leaving group during coupling to serine , lose a second group in a process called aging . the rate of aging is fast for soman ( half - life 2 min ) and relatively slow for sarin ( half - life of 6 h ) bound to wild - type bche . soman - inhibited atypical bche regained 16% , whereas soman - inhibited wild - type bche regained 7% of its original activity following incubation with hi-6 oxime . this result can be interpreted to mean that soman bound to atypical bche ages more slowly . a parallel study for soman - inhibited human ache showed that the d74n mutant , homologous to atypical bche , aged 15-fold more slowly than soman - inhibited wild - type ache . the rate of aging of paraoxon - inhibited atypical bche is 3-fold slower than the rate for paraoxon - inhibited wild - type bche and 8-fold slower for the diisopropyl fluorophosphate adduct . aged adducts are irreversibly inhibited , but unaged adducts can undergo spontaneous or oxime assisted reactivation . the atypical and k - variant have decreased levels of bche molecules in plasma , calculated from antibody - based assays . the concentration of wild - type bche in plasma is 4 mg / l ( 50 nm ) , while the concentration of homozygous atypical bche in plasma is 70% of wild - type bche . the homozygous k - variant has 33% fewer bche molecules per ml plasma compared to wild - type bche . silent bche variants resulting from frame shift , splice junction mutations , or stop codon insertions are associated with complete absence of bche molecules , while silent variants resulting from missense mutations can have the protein but no bche activity . fewer active bche molecules means reduced bioscavenger capacity , resulting in a larger dose of toxicant reaching the neuromuscular junction . inhibition of ache in the neuromuscular junction causes muscle weakness and respiratory failure . in conclusion in contrast , atypical bche is an inefficient bioscavenger of positively charged cholinesterase inhibitors . individuals who carry the atypical bche variant may suffer toxic effects from low doses of vx or neostigmine that cause no toxicity in persons with wild - type bche . genetic variants with abnormally low levels of bche enzyme including the atypical , k - variant , and silent variants are predicted to be unusually sensitive to toxicity from low doses of nerve agents . only a few reports determined bche genotype in individuals intoxicated by cholinesterase inhibitors . one israeli soldier , homozygous for atypical bche , suffered severe cholinergic symptoms following pyridostigmine prophylaxis during the 1991 persian gulf war . a case - control study evaluated 144 veterans who had gulf war illness and 160 veterans who also served in the persian gulf war but had no symptoms . veterans used pyridostigmine bromide during the gulf war as a protective measure against nerve agents . they also used cholinesterase - inhibiting pesticides and were possibly exposed to low levels of sarin and cyclosarin following demolition of an iraqi compound near khamisiyah . gulf war illness risk was dramatically elevated for veterans with the less common bche genotypes who reported taking pyridostigmine bromide pills during deployment . brazilian farmers who had no signs of poisoning but who treated their crops with organophosphorus and carbamate pesticides were screened for relative erythrocyte ache levels . out of 134 farmers , the less common bche genotypes were more frequent in the mildly poisoned group than in the control group . it was concluded that individuals with nonusual bche phenotypes seem to be predisposed to erythrocyte ache inhibition . whereas wild - type bche protects ache from inhibition by destroying pesticides , bche variants are less efficient detoxifiers . no signs of toxicity were seen in human volunteers administered chlorpyrifos orally at doses that inhibited plasma bche activity 85% . plasma bche activity is considered a biomarker of exposure but is an inappropriate end point for risk assessment . animal studies indicate that developmental effects on the fetus occur at chlorpyrifos doses that produce maternal toxicity and inhibit erythrocyte ache activity but not at low doses that produce no maternal toxicity and no erythrocyte ache inhibition . these studies confirm that bche is highly reactive with chlorpyrifos oxon , the toxic metabolite of chlorpyrifos , whereas ache is resistant to inhibition , their rate constants being 3.8 10 m min for human ache and 1.6 10 m min for human bche . the 3-orders of magnitude difference in chlorpyrifos oxon reactivity supports the conclusion that the toxic dose is much higher than the 50 nm concentration of plasma bche , indicating that 50 nm bche contributes little to no protection from toxicity . individuals with genetic variants of bche are not expected to be unusuallly sensitive to op toxicity . howard et al . formulated a new approach to evaluate the risk of toxicity from op by studying genetic variability in 30 genes involved in op metabolism . gomez - martin built on this idea by analyzing 10 polymorphic variants of seven genes involved in organophosphorus pesticide metabolism . the goal was to identify individuals most at risk of adverse health effects from exposure . they defined risk as mutations in butyrylcholinesterase ( bche ) , paraoxonase-1 ( pon1 ) , and cytochrome p450 enzymes ( cyp2d6 , cyp2c19 , and cyp3a ) and copy number variation in glutathione s - transferases ( gstm1 and gstt1 ) . out of 496 individuals , 172 ( 34.7% ) carried an allele for the k - variant or atypical bche , while 58 ( 11.7% ) presented the pon1 genotype for slow metabolism of op pesticides . null genotypes for gstm1 and gstt1 were found in 7.1% of the study population . when genetic polymorphisms in bche , pon1 , gstm1 , and gstt1 were considered together , the worst genotype combination was observed in one individual ( 0.2% ) . when cyp450 enzymes were assessed for the worst genotype combinations , only 2 indivduals carried risky genotypes , cyp3ap1 - 44gg and cyp2c19 681aa , which correspond to extensive and poor metabolizers , respectively . the individuals in the study had no toxic symptoms and no known exposure to op . greenhouse workers had higher levels of pon1 activity than the control group suggesting an adaptive response to pesticide exposure . greenhouse workers also had higher levels of bche activity but only when exposure levels were low . several esterases with phenyl valerate activity in chicken brain are inhibited by op including neuropathy target esterase and bche . the human studies of healthy young men summarized in section 3.1 document the fact that individuals respond differently to the same dose of nerve agent administered via the same route ( iv or orally ) . at the time of the studies , methods for identifying genetic variants polymorphism of bche and op - metabolizing genes is a likely explanation for why some people have toxic symptoms from low dose exposures that have little effect on others . the response of nonhuman primates to op toxicants is closest to that of humans . sequencing of the rhesus macaque exomes identified a family of monkeys with a naturally occurring mutation p.gly180 * in the bche gene in the heterozygous state . the nonsense mutation introduced a stop codon , resulting in a decrease of serum bche activity . monkeys with this mutation could serve as a model for humans with bche mutations in the heterozygous state to determine whether bche deficiency is associated with increased sensitivity to op toxicity . directed breeding of heterozygous monkeys with this mutation could be used to produce homozygous animals for study of the effects of complete absence of bche in a nonhuman primate . the bche knockout mouse with no bche activity in any tissue is not an appropriate model for the effect of soman in humans who are deficient in bche because the mice have high levels of carboxylesterase activity in plasma . humans have no carboxylesterase in blood . mouse plasma carboxylesterase , being an efficient bioscavenger of soman , obscures the contribution of bche . mice with no plasma carboxylesterase , but with normal carboxylesterase in other organs , are a more suitable model for human response to nerve agents . their bche activity per ml plasma is less than 50% of the bche activity in human plasma . a low dose of the nerve agent analogue , soman coumarin , was lethal to es1/ mice but not to es1+/+ mice , demonstrating that plasma carboxylesterase protects from a highly toxic soman analogue . the mice can be purchased from the jackson laboratory , bar harbor , me catalog # b6(cg)-ces1c / j stock no : 014096 . studies in plasma carboxylesterase knockout mice demonstrated that carboxylesterase does not protect from op pesticides . the explanation for the failure of plasma carboxylesterase and bche to protect from op pesticides is as follows . op pesticides react poorly with ache and therefore cause toxicity only at high doses . a toxic dose of op pesticide exceeds the concentration of bioscavenger in plasma . even though the op pesticide is effeciently scavenged in plasma , all of the scavenging capacity is used up before a significant amount of the op is destroyed , thereby affording no protection . paraoxonase ( pon1 ) in human plasma is a component of high density lipoprotein . pon1 hydrolyzes the active metabolites of op pesticides including chlorpyrifos oxon and diazoxon , as well as the nerve agents sarin and soman . mice completely deficient in pon1 are highly sensitive to the toxicity of chlorpyrifos oxon and diazoxon . pon1 knockout mice can be purchased from the jackson laboratory b6.129 1-pon1/j stock no : 004160 . the atypical and k - variant have about 70% of the standard concentration of bche molecules per ml plasma . a previously reported mass spectrometry method can detect as little as 3.2% nerve agent - labeled bche in 0.5 ml of plasma . this high level of sensitivity means that the atypical and k - variants of bche are expected to yield detectable levels of bche adducts . silent variants range from zero activity and no bche protein , to 10% of normal activity and 50 nm bche protein . the silent bche variants with no bche protein will yield no bche op adducts . the missense mutation that changes the active site serine to glycine makes a bche protein that is unable to make a covalent bond with op . plasma from individuals with these bche mutations will give no information about op exposure in the standard mass spectrometry assay . the quantity of soluble ache in plasma ( 8 ng / ml ) is 500- to 1000-fold lower than that of bche ( 4000 ng / ml ) . rbc are a richer source of ache than plasma , but analysis of ache on rbc requires additional sample preparation steps because ache is membrane bound . higher than normal bche activity , ranging from 30% to 400% of the normal , is found in the cynthiana and c5 + genetic variants . these variants have the wild - type bche nucleotide sequence in their exons and promoter region . heritable quantitative trait loci have been identified in intron 2 and the 3-utr of the bche gene and on chromosome 5q , but it is unknown whether mutations at these loci increase or decrease bche activity levels . additional loci that affect bche activity have been identified by the genome - wide association scan method . the frequency of the c5 + variant is 10% in european and american populations . plasma samples from these individuals are expected to give strong signals in previously reported mass spectrometry assays for confirming op exposure . ache genetic variants have not been associated with the risk of toxicity from cholinesterase inhibitors . the most frequent variant his353asn ( h322n ) , the yt blood group antigen , has normal ache activity . missense mutations are rare , and when they do occur , they are paired with a normal ache allele . some diseases are associated with very low ache enzyme activity , but the cause is not a mutation in ache . congenital myasthenic syndrome with end - plate acetylcholinesterase deficiency is caused by mutations in the colq gene , resulting in failure to anchor ache to the neuromuscular junction . patients with paroxysmal nocturnal hemoglobinuria have a deficiency in all glycolipid - anchored proteins including ache , explained by mutations in enzymes that synthesize the glycosylphosphatidylinositol anchor . it is predicted , but unproven , that patients with ache deficiency have an increased risk of toxicity from cholinesterase inhibitors . it is commonly assumed that people with bche deficiency are at increased risk of toxicity from cholinesterase inhibitors . in our opinion , this generalization is correct for nerve agents but fails for cholinesterase inhibitors that have a higher affinity for bche than ache . as pointed out by maxwell and confirmed by studies in carboxylesterase knockout mice , poisons that preferentially react with plasma bioscavengers rather than with ache produce no toxic signs until the bioscavenger capacity is exceeded . the binding affinity of op pesticides for ache is so poor that ache is inhibited only by large doses . toxic doses of op pesticides are orders of magnitude higher than the bche concentration in blood . the 50 nm bche in human plasma scavenges 50 nm op pesticides but is unable to scavenge 20,000 nm chlorpyrifos . thus , bche contributes little to no protection from the acute toxicity of op pesticides . this means that risk from op pesticide toxicity is indistinguishable in individuals with genetic variants from those with wild - type bche . humans have moderate signs of toxicity after a single oral dose of 0.028 mg / kg sarin or an intravenous dose of 0.0015 mg / kg vx . the 50 nm bche in plasma easily captures low doses of nerve agent , preventing the inhibition of ache . genetic variants with reduced levels or with inactive bche are expected to be at increased risk of toxicity from nerve agents . this prediction assumes that detoxication of nerve agents is accomplished primarily by bche . it does not consider the role of other bioscavengers including the carboxylesterases in the liver and lung , acetylcholinesterase in rbc , albumin in plasma , metabolic destruction by cytochrome p450 and paraoxonase , glucuronide conjugation , and glutathione - mediated conjugation . body fat also has a role because the uncharged cholinesterase inhibitors are sequestered in body fat . what is needed is a study that correlates the toxicity of nerve agents with bche genotype . if archived blood samples exist from humans administered known doses of sarin and vx , bche genotype analysis would answer the question of whether bche genetic variants explain the differences in toxic response noted in individual subjects .
acetylcholinesterase ( ache ) is the physiologically important target for organophosphorus toxicants ( op ) including nerve agents and pesticides . butyrylcholinesterase ( bche ) in blood serves as a bioscavenger that protects ache in nerve synapses from inhibition by op . mass spectrometry methods can detect exposure to op by measuring adducts on the active site serine of plasma bche . genetic variants of human ache and bche do exist , but loss of function mutations have been identified only in the bche gene . the most common ache variant , his353asn ( h322n ) , also known as the yt blood group antigen , has normal ache activity . the most common bche variant , ala567thr ( a539 t ) or the k - variant in honor of werner kalow , has 33% reduced plasma bche activity . the genetic variant most frequently associated with prolonged response to muscle relaxants , asp98gly ( d70 g ) or atypical bche , has reduced activity and reduced enzyme concentration . early studies in young , healthy males , performed at a time when it was legal to test nerve agents in humans , showed that individuals responded differently to the same low dose of sarin with toxic symptoms ranging in severity from minimal to moderate . additionally , animal studies indicated that bche protects from toxicants that have a higher reactivity with ache than with bche ( e.g. , nerve agents ) but not from toxicants that have a higher reactivity with bche than with ache ( e.g. , op pesticides ) . as a corollary , we hypothesize that individuals with genetic variants of bche may be at increased risk of toxicity from nerve agents but not from op pesticides .
You are an expert at summarizing long articles. Proceed to summarize the following text: acute promyelocytic leukemia ( apml ) is a distinct subtype of aml with specific clinical , morphologic , and genetic features which is treated differently than other aml subtypes . patients in the low - risk group ( platelet counts > 40 109/l and wbc count < 10 109/l ) and intermediate risk group ( platelet counts < 40 109/l and wbc count < 10 109/l ) rarely experience relapse ( < 3% ) , whereas patients in the high - risk group ( wbc counts of greater than 10 10/l ) are reported to have a relapse rate of 2840% 1 . molecular monitoring of the minimal residual disease ( mrd ) by qualitative nested reverse transcription polymerase chain reaction ( rt - pcr ) and quantitative real - time pcr of promyelocytic leukemia / retinoic acid receptor- ( pml / rar ) allows to follow the individual kinetics of mrd and to identify patients with an imminent hematological relapse . till now , multiple case reports of lineage switch from acute lymphoblastic leukemia ( all ) to aml and few reporting vice versa have been published 2,3 . herein , we report a case of apml which showed switch to aml - non - m3 after 2 years of arsenic and all - trans retinoic acid ( atra ) treatment . to the best of our knowledge , this is the first case of apml showing phenotypic switch to a aml - non - m3 and second case of switch between aml french american british ( fab ) classification subgroups . a 5-year - old girl presented with fever , neck swelling , and weight loss for 2 months in april 2011 for which she was started on antitubercular drugs by a local physician . on examination , she had pallor , bilateral cervical lymphadenopathy , and hepatosplenomegaly ( liver 3 cm and spleen 3 cm below costal margin ) . the hemoglobin was 71 gm / l , total leukocyte count was 8.1 10/l , and platelet count was 31 10/l . d - dimer test , prothrombin time ( pt ) , and activated partial thromboplastin time ( aptt ) were within normal limits . peripheral blood smear showed 80% abnormal promyelocytes which were immunopositive for cmpo , cd45 , cd33 , cd117 , cd2 , cd13 ( dim ) , and cd38 ( dim ) and immunonegative for cd34 , hladr , ccd79a , cd19 , cd18 , cd4 , cd7 , and ccd3 . bone marrow ( bm ) aspiration revealed near total replacement by abnormal promyelocytes ( fig.1 ) . the patient was started on arsenic monotherapy ( 0.15 mg / kg per day ) and after 60 days of induction , morphological remission in bm and absence of pml / rar transcripts on rt - pcr were reported . induction was followed by one cycle of consolidation of 28 days with arsenic ( 0.15 mg / kg per day ) and six cycles of maintenance ; monthly cycle of arsenic for 10 days / month ( 0.15 mg / kg per day ) and intrathecal methotrexate on day 1 ( 8 mg ) till march 2012 . the child was apparently well for 1 month , after which she again presented with fever . five percent abnormal promyelocyteson peripheral blood smear and presence of pml / rar transcripts on rt - pcr were noted . at this time , treatment was initiated with atra ( 45 mg / m per day ) and daunomycin ( 1.5 mg / kg per day ) . morphological remission in bm was reported after completion of 3 months of induction and thereafter consolidation with atra ( 0.15 mg / kg per day ) , daunorubicin , and cytarabine on day 1 . within 2 months of consolidation phase , patient relapsed again and the bm at this time was replaced by blasts which were immunopositive for cmpo , cd45 , cd34 , cd13 , cd33 , cd117 , cd15 , cd11b , cd56 , cd19 , cd64 , cd2 , cd65 , cd7 , hladr , cd9 , cd4 , and cd133 and negative for cd38 , ccd79a , and ccd3 , suggestive of aml - non - m3 ( fig.2 ) . a diagnosis of switch to aml - non - m3 from aml - m3 was made . the prognosis was discussed with the family and the child was started on medical research council 's 10th aml ( mrc aml10 ) protocol . the child succumbed to death after 2 months due to sepsis ( febrile neutropenia ) , refractory shock , and ongoing myocardial dysfunction for which the child was on cardioprotective drugs . peripheral blood smear showing abnormal promyelocytes at diagnosis ( jenner and giemsa , 1000 ) . peripheral blood smear showing abnormal myeloblasts ( jenner and giemsa , 1000 ) . a lineage switch phenomenon is observed when acute leukemia that meet the standard fab criteria for a lineage ( lymphoid or myeloid ) at the time of initial diagnosis meet the criteria for opposite lineage at the time of relapse 4 . most of the lineage switch cases are associated with poor outcome 2 the bad prognostic factors being congenital acute leukemia ( cal ) 57 and chromosomal aberrations including rearrangements involving mll gene ( 11q gene ) 8,9 . conversion of aml to all or vice versa does not seem to have a role in prognosis 3,4 . the case described earlier can be considered as phenotypic switch as leukemic clones have switched to different phenotype within the same lineage . hematopoietic system consists of progenitor stem cells that gradually lose multiple alternate potentials to commit to some lineage fates . multiple potential mechanisms of lineage switch in acute leukemia in a nonhematological stem cell transplant case are proposed . the presence of early bipotential b - macrophage progenitors in the bm and the fact that mll - positive b - all show gene expression profiles consistent with early hematopoietic progenitors have raised the possibility that early bipotential or oligopotential progenitor cells are target for leukemogenic translocations , and constitute the origin of lineage switching events 10 . another possibility is that the ectopic expression or deletion of master regulators ( such as runx1 , scl , ikaros , gfi1 pu.1 , gfi1 , c / ebp , c / ebf , pu.1 , and gata-1 required for the commitment of myeloid cells , and notch1 , gata3 , and pax5 , which mediate t- and b - cell development ) mostly result in lineage reprograming with or without reversionof cells back to a multipotent stage which is potentiated in a changed microenvironment of the bm 11,12 . third possibility is that chemotherapy might supress or eradicate the leukemic clone that is apparent at the time of diagnosis , thereby permitting the expansion of a clone with a different phenotype 13 . a review article in 2012 on lineage switch describes 18 pediatric case reports of lineage switch mostly occuring from all to aml - m4/m5 and vice versa mostly . why specifically most lineage switch occurs between aml - m4/m5 regarding phenotypic switch in aml between various subgroups , till now only one case report in 2002 has been published regarding switch from aml - m0 to aml - m7 on relapse 14 . coming to our case which has been the switching from aml - m3 to aml - non - m3 , there was no evidence of mll / bilineage / biphenotypic markers at the time of diagnosis , no evidence of clonal change at the time of relapse , and no stem cell transplantation has been done . other rare gene arrangements conferring poor prognosis in apml - like stat 5b and numa could not be tested at diagnosis due to nonavailability of the same in our institution and poor financial status of the patient . against a second malignancy , points are that atra and dnr used for apml treatment in this patient are not leukemogenic and the child had not yet been declared cured from the treatment when this switch was noted . as such case has not been published till date and required genetic markers for understanding the mechanism of phenotypic switch could not be done in the described case , new management strategies could not be entertained . in conclusion , though our current findings are insufficient to confirm the mechanism of switch from aml - m3 to aml - non - m3 and as it has been not previously documented , till now , it can be said that it is due to cellular reprograming and redifferentiation to common myeloid precursors and thereafter aml - non - m3 in a favorable microenvironment of bm modified to some extent by chemotherapeutic agents used for treatment .
key clinical messagephenotypic switch between any leukemia subtypes is of concern to a treating physician and more so , in acute myelocytic leukemia ( aml ) as the mechanisms for switch and subsequent chemotherapy regimen to be used remain unclear . aml - non - m3 from aml - m3 subtype needs special mention as this has been unheard off .
You are an expert at summarizing long articles. Proceed to summarize the following text: chemotherapy remains the main treatment option for most cancers despite of its limitations , such as systemic toxicity , severe side effects , and limited efficacy . the major reason for chemotherapy failure is poor delivery of drug to cancer cells and/or intracellular targets . there are a number of barriers that have to be overcome for successful treatment , and multidrug resistance ( mdr ) is one of them . tumors of different origin have different susceptibility to chemotherapy , and frequently cancers are intrinsically resistant . on the other hand , even though many primary tumors and metastatic lesions , for example breast , ovarian , and small cell lung carcinomas initially respond well to the chemotherapeutic treatment , cancers often relapse and develop drug resistance . moreover , cancer cells simultaneously acquire resistance not only to the drug the patient was treated with but also to the broad spectrum of drugs that are structurally and functionally unrelated to each other . initially mdr was attributed to the expression of drug efflux transporters on the cell membrane that actively pump the drugs out of the cells . now it is generally recognized that mdr is a complex phenomenon and usually is governed by one or more of the following mechanisms : ( 1 ) active drug removal by drug efflux transporters of the atp - binding cassette ( abc ) superfamily , such as p - glycoprotein ( pgp , abcb1 ) , multidrug resistance - associated protein 1 ( mrp1 , abcc1 ) , and breast cancer resistance protein ( bcrp , abcg2 ) ; ( 2 ) loss of cell surface receptors or drug transporters or alterations in membrane lipid composition that limit diffusion of the drug into the cells ; ( 3 ) compartmentalization of the drug in cellular vesicles ; ( 4 ) altered / increased drug targets ; ( 5 ) increased drug metabolism ; ( 6 ) alterations in cell cycle ; ( 7 ) active damage repair ; and ( 8) inhibition of apoptosis ( figure 1 ) . mechanisms of mdr in cancer cells : ( 1 ) active drug efflux by drug transporters , such as pgp , mrp , and bcrp ; ( 2 ) loss of cell surface receptors and/or drug transporters or alterations in membrane lipid composition ; ( 3 ) compartmentalization of the drug in cellular vesicles ; ( 4 ) altered / increased drug targets ; ( 5 ) alterations in cell cycle ; ( 6 ) increased drug metabolism / enzymatic inactivation ; ( 7 ) active damage repair ; and ( 8) inhibition of apoptosis . despite much effort contributed to overcoming mdr , the success is still very limited in clinical settings . first , the modification of treatment regimens by increasing the dose of the administered drug(s ) or using non - cross - resistant drugs . second , use of small molecule inhibitors of drug efflux transporters to increase the drug uptake in mdr tumors . third , use of antibodies and antibody fragments to target and inhibit drug efflux transporters . fourth , silencing of the gene expression of the drug efflux transporters or antiapoptotic proteins , such as bcl2 using antisense oligonucleotides , sirna , or micro rna . fifth , use of small molecules to suppress non - abc transporter - mediated resistance . finally , use of nanotechnology - based carriers to bypass drug efflux transporters in mdr cancer cells . of unfortunately , a simple dose increase has been associated with increased risks of systemic toxicity and severe side effects , while finding a proper combination of non - cross - resistant drugs in many cases is complicated . as far as the use of the pgp inhibitors is concerned , the outcomes were often poor , and many such inhibitors failed due to toxicity or drug metabolism associated issues . moreover , most of the approaches under development face traditional drug delivery issues , which are especially severe in the cases of nucleic acid or protein therapeutics . nanotechnology offers several advantages both for the delivery of the chemotherapeutic agents , allowing them to bypass drug efflux transporters , and for the delivery of agents that could inhibit drug resistance mechanisms to increase efficacy of the chemotherapy . nanomedicines have longer circulation times and can passively accumulate in the tumors with leaky vasculature and poor lymphatic drainage by the enhanced permeability and retention ( epr ) effect . attaching specific tumor - targeting antibodies , antibody fragments , or other targeting moieties ( receptor ligands , peptides , etc . ) can result in active targeting of the nanomedicines to the tumor cells , which can further improve drug delivery . second , two or more active compounds can be incorporated into a single carrier allowing simultaneous delivery of several cytotoxic drugs for combination therapy and/or a cytotoxic drug with a mdr modulator , such as small molecule inhibitor , antibody , or nucleic acid . third , a nanocarrier can be designed in such a way that it will release its cargo at the tumor site in response to specific tumor conditions , such as ph or presence of particular enzymes , therefore limiting other organs and tissues to the exposure to free drug and reducing systemic toxicity . finally , in contrast to small molecules that mainly utilize diffusion to penetrate the cells , nanocarriers are taken up by either passive endocytosis or receptor - mediated endocytosis and , therefore , can bypass drug efflux transporters on the plasma membrane . in the latter case the endocytosis is triggered by interaction of targeting ligand with its receptor on plasma membrane , which accelerates the uptake compared to passive endocytosis . if the receptor is predominantly expressed on cancer cells , in addition to faster uptake this allows selective targeting of the nanocarrier to cancer cells . one such example is represented by a class of copolymers , called pluronic block copolymers or poloxamers , that are widely used in various drug delivery systems and in tissue engineering . pluronics are triblock copolymers of poly(ethylene oxide ) ( peo ) and poly(propylene oxide ) ( ppo ) , arranged in peo - ppo - peo structure . depending on the length of the blocks the hydrophilic lipophilic balance ( hlb ) of the copolymers changes . in the solution pluronics the core of the micelles contains ppo blocks and allows incorporation of hydrophobic drugs . previously thought to be inert , pluronics display a unique set of biological activities and have been shown to be potent sensitizers of mdr cancer cells in vitro and in vivo . moreover , pluronics were shown to prevent the development of mdr upon selection with an anthracycline antibiotic , doxorubicin ( dox ) , both in vitro and in vivo . we have also recently demonstrated that pluronics in combination with dox can deplete tumorigenic cell subpopulations and decrease cancer cells tumorigenicity and tumor aggressiveness upon treatment in vivo . in this review the first drug efflux transporter in cancer cells was described by juliano and ling in 1976 . they have shown that drug - resistant chinese hamster ovary cells express a 170 kda membrane glycoprotein , now known as p - glycoprotein ( pgp , abcb1 ) , that was unique to the drug - resistant cells . the cells were selected for resistance to colchicine and showed cross - resistance to a wide range of different compounds . the degree of drug resistance correlated with the amount of pgp on the cell surface . later , in early 1990s a second drug efflux transporter , called multidrug resistance - associated protein ( mrp1 or abcc1 ) , was discovered in a drug - resistant lung cancer cell line . normally mrp1 plays a major role in cell detoxifying mechanism by transport of exogenous and endogenous compounds conjugated to glutathione ( gsh ) , which for some substrates is required as a cofactor for mrp1 activity . in contrast , pgp does not require a cofactor and can efflux a wide variety of functionally and structurally diverse but commonly hydrophobic drugs . another important drug efflux transporter , named breast cancer resistance protein ( bcrp , abcg2 ) , was identified in 1998 by doyle et al . in human breast cancer cell line selected for dox resistance . its expression is associated with resistance to number of drugs , such as mitoxantrone , camptothecins , anthracyclines , etc . pgp , mrp1 , and bcrp belong to the large superfamily of atp - binding cassette ( abc ) membrane transporters with 48 members of the superfamily that are divided into 7 subgroups ( a g ) . they have conserved structures and ubiquitously expressed in all forms of living organisms , from bacteria to humans . it is a product of mdr1 gene and can be found in many normal tissues , like epithelial cells of gastrointestinal tract , liver , the luminal membrane of proximal tubular epithelial cells in kidney , cornea , and the luminal membrane of the endothelial cells in the blood overall , pgp is mostly expressed in tissues with barrier functions and its main role is to protect the organism from toxic compounds . it has a typical structure for abc transporters and comprises two trans - membrane domains ( tmds ) , each of which has 6 membrane - spanning helixes , and two intracellular nucleotide - binding domains ( nbds ) , which bind and hydrolyze atp providing energy for transmembrane movement of the drugs ( figure 2 ) . pgp substrates are mostly hydrophobic ( but structurally unrelated ) and partition into a lipid bilayer . among these substrates are important anticancer drugs including several anthracyclines ( dox , daunorubicin , mitoxantrone ) , vincristine , taxanes , etoposide , teniposide , actinomycin d , and others . understanding the mechanism of pgp function is critical for the design of novel effective mdr modulators . recently the crystal structure of mouse pgp , which has 87% sequence identity to human pgp , was described ( figure 2 ) . by analyzing the costructures of pgp complexes with two cyclopeptide inhibitors the authors elucidated the mechanism of drug efflux by pgp and provided insight into the transporter s broad substrate specificity . the drug - binding pocket of pgp is localized in the tm domain of the protein . the inward - open conformation of pgp allows the substrate access both from cytoplasm and from the inner leaflet of the membrane but not from the upper leaflet or extracellular space . the upper part of the drug - binding pocket contains predominantly hydrophobic and aromatic amino acid residues , and the lower half of the chamber has more polar and charged residues . the drug - binding pocket in pgp is very large and in inward - facing conformation is accessible through two portals that are wide enough to fit hydrophobic drugs and phospholipids and allow pgp to scan the inner leaflet to select and bind specific lipids and hydrophobic drugs before transport . overall , the authors proposed , that pgp has broad flexibility and can sample widely open conformations to accommodate large substrates , explaining the broad substrate specificity of the transporter . usually the drug enters pgp s binding site from the inner leaflet of the membrane , which stimulates the binding of two molecules of atp by nbds followed by their dimerization . the dimerization of nbds causes the major conformational change in the protein and formation of the outward - facing structure , open to the extracellular space . the drug is released due to the change of the affinity of the protein to it or is facilitated by atp hydrolysis , which brings the protein back to the initial state . ( a ) pgp is a transmembrane protein with drug - binding pocket localized in the inner leaflet of the plasma membrane , and two nbd localized in cytoplasm . functional pgp is localized in cholesterol , sphingomyelin , and gm1 ganglioside - rich membrane microdomains , called lipid rafts , where it is surrounded by fluid phase of the membrane , containing unsaturated fatty acids like dppc . pgp is pictured in inward - open ( outward closed ) conformation ready to bind substrate . the model is based on x - ray analysis and nmr data from protein data bank ( http://www.rcsb.org/ ) . ( b ) incorporation of pluronic into lipid bilayer disrupts lipid rafts , possibly causing conformational changes in pgp , which results in inhibition of pgp atpase and transport activities . as was mentioned above , pluronic block copolymers are potent sensitizers of mdr cells . the sensitization mechanism is complex and involves multiple events happening at different levels in the cell . cell interaction starts in the cell membrane , where drug efflux transporters are localized . pluronics were shown to be strong inhibitors of abc transporters , specifically pgp , mrp , and bcrp . the inhibition might be in part due to the alterations of lipid microenvironment of the transporters by pluronic . due to their amphiphilic structure , pluronic block copolymers can interact with cell membrane and change its properties , which are critical for proper function of abc transporters . membrane structure and composition play a crucial role in cell physiology , function , and signaling . plasma membrane is a heterogeneous structure composed of various domains with different lipid composition and packing . in particular , so - called lipid rafts are compact membrane microdomains containing predominantly cholesterol and sphingolipids ( mainly sphingomyelin ) with long and saturated fatty acids , that are floating in more fluid membrane phase that contains glycerophospholipids with shorter and unsaturated acyl chains ( figure 2 ) . these domains are resistant to low temperature solubilization by some detergents , like triton x100 or brij 96 , and this is used for their isolation . depending on the cell line and the method used for membrane fractionation pgp can be found either mostly in detergent - resistant membrane fractions or distributed between the detergent - resistant and detergent - soluble fractions . furthermore , it was found that pgp distribution between different membrane fractions depends on the transporter s expression level : the lower the expression of pgp is , the greater portion of pgp is localized in detergent - resistant cholesterol - rich membrane domains . it is well - known that the function of most membrane proteins is directly linked to the composition and viscosity of their lipid microenvironment . pgp is a lipid flippase and requires interaction with phospholipids for continuous display drug - mediated atpase activity and interaction with the substrate . moreover , an increasing number of studies report that pgp localization in lipid rafts and precise properties of rafts are essential for the transporter s proper function . for example depletion of cholesterol with methyl--cyclodextrin in drug - resistant vlb human t - cell lymphoblastic leukemia cells led to disassembly of the lipid rafts , redistribution of pgp from lipid rafts to other microdomains of plasma membrane , and inhibition of pgp transporter activity . on the other hand , enrichment of membranes with cholesterol also resulted in inhibition of pgp function , although the localization of pgp did not change compared to control . however , the increase in cholesterol content changed the lipid raft distribution and composition , which most likely accounts for the impairment of the pgp function . it was also shown recently that caveolin-1 overexpression decreases plasma membrane cholesterol levels ( similar to the effect of methyl--cyclodextrin that depletes cholesterol from the membrane ) and results in the increase of membrane fluidity and inhibition of pgp function in drug - resistant hs578t / dox cells . another study by barakat et al . demonstrated that there are two functionally different populations of pgp in drug - resistant human cem lymphoblastic leukemia cells . the first population localized in detergent - resistant membrane fraction has higher atpase activity , which is completely inhibited by orthovanadate and activated by verapamil . the second population localized in soluble membrane fractions has lower atpase activity and is less sensitive to orthovanadate . moreover , verapamil , a well - known pgp activator , inhibits pgp atpase activity in this second population . the authors conclude that interaction of pgp with its substrates could be affected by different lipid microenvironment in soluble membrane fractions , specifically by lower content of cholesterol compared to the detergent - resistant membrane fraction . pluronic binding to the cell membrane depends on pluronic hydrophobicity and the temperature . the binding is driven by hydrophobic interactions of ppo chain blocks with the fatty acid residues in the lipid bilayer and by hydrophilic interactions of peo chain blocks with the polar groups of the lipids at the membrane surface . this binding may lead to either membrane destabilization or healing of injured membranes . pluronics also exhibit ionophoric activity and can facilitate transmembrane transport of low molecular drugs , accelerate phospholipid s flip - flop rate , and decrease membrane microviscosity . pluronic effects on the membrane transport depend on the copolymer hlb , concentration , and the exposure time . for example , hydrophobic pluronic l61 ( ( eo)4-(po)30-(eo)4 , hlb 3 , mw 2000 g / mol , eo = ethylene oxide ; po = propylene oxide ) depending on the level of its aggregation can act either as a transmembrane carrier of drug molecules or as an ion channel . specifically , it was proposed that l61 monomers and dimers can act as the carriers while l61 oligomers are likely to form the channels . on the other hand hydrophilic pluronic f68 ( poloxamer 188 , ( eo)76-(po)39-(eo)76 , hlb 29 , mw 8400 g / mol ) with 80% peo content effectively restores damaged cell membranes after electroporation , heat shock , or intense radiation . using x - ray reflection ( xr ) and grazing - incidence x - ray diffraction ( gixd ) methods in a model langmuir lipid monolayer of 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ( dppc ) and 1,2-dipalmitoyl - sn - glycero-3-[phospho - rac-(1-glycerol ) ] ( dppg ) , wu et al . have shown that f68 interacts with the damaged membrane areas , but does not affect the ordered membrane phase , and gets excluded when lipid packing density is restored . recently it was demonstrated that f68 molecules do not insert into lipid bilayer nor affect the overall lipid packaging , however , they facilitate the membrane sealing activity by diminishing the fluctuation of membrane surface and hydration of the inner part of the bilayer . however , in another study using giant unilamellar vesicles ( guv ) as model membrane system wang et al . demonstrated that f68 can incorporate in the membranes , disrupt their integrity , and act as a permeabilizer if it is exposed to the membranes for sufficient time . overall , the interaction of pluronics with lipid membranes proceeds in two steps : ( 1 ) the absorption at the membrane and ( 2 ) the insertion in the membrane ( figure 3 ) . the first step is common to all pluronics and does not depend much on the copolymer structure . the second step depends strongly on the hydrophobicity of the copolymer with the more hydrophobic copolymers being morel likely to insert . pluronics with longer ppo blocks insert into the membrane below the polar head groups , loosen the lipid packaging , and , therefore , act as permeabilizers , they can translocate through the membrane ( depending on their hlb ) . furthermore , using molecular dynamics simulations nawaz and coauthors observed that membrane bends upon insertion of pluronics . they have shown that membrane - disruptive activity of pluronics is due to interaction of hydrophilic blocks with the polar head groups of the lipid molecules and depends on the length of the peo block . short peo blocks drag the polar groups toward the inner part of the membrane , which results in membrane bending and permeabilization . pluronics with longer peo blocks can temporarily stabilize the local structure of the membrane . schematic presentation of interaction of pluronics with different hydrophobicity with lipid membranes : ( 1 ) absorption of pluronic molecules on the surface of the membrane , ( 2 ) insertion into the lipid bilayer , and ( 3 ) translocation through the membrane . pluronic copolymers can significantly increase the antitumor activity of pegylated liposomal drugs in vivo , specifically doxil by stimulating the drug release from liposomes at the tumor site . one of the main problems of long circulating liposomal drugs is insufficient release of the active compound at the tumor site . post - administration of pluronic p85 ( ( eo)26-(po)40-(eo)26 , hlb 16 , mw 4600 g / mol ) 48 h after doxil results in dox release and redistribution toward tumor bulk along with a marked improvement of antitumor activity . this effect is time - dependent as it is essential to allow sufficient time for the liposomes to accumulate at the tumor site before administering pluronic . it is likely that that the enhanced antitumor effect at least in part is due to facilitated release of dox from the liposomes in the tumors induced by pluronic . furthermore , in addition to permeabilization effect on liposomal membranes the copolymer could also sensitize the mdr cells and deplete the cancer stem cells ( cscs ) ( as discussed below ) . another important aspect in pluronic interactions with lipid membranes is the dependence of these interactions on the cell type and the membrane composition . for example , the membrane microviscosity of murine myeloma sp2/0 cells significantly decreased after treatment with l61 , while the membrane viscosity in normal mouse splenocytes was less affected . moreover , pluronic p105 ( ( eo)37-(po)56-(eo)37 , hlb 15 , mw 6500 g / mol ) was demonstrated to permeabilize the acidic endosomal vesicles in drug - resistant a2780/adr cells , while the vesicles in sensitive cells were less affected . several studies have reported lower fluidity and higher heterogeneity of plasma membrane in mdr cells compared to sensitive cells . drug - resistant cells also contain smaller amounts of unsaturated fatty acids and have higher content of esterified cholesterol and triglycerides . using liposomes of different lipid composition and viscosity it was demonstrated that the l61 effects on lipid flip - flop and membrane permeability toward dox increase as the membrane viscosity increases . pluronics inhibit pgp and mrp atpase activities by decreasing maximum reaction rate ( vmax ) and the affinity of the enzyme to atp as well as to the substrates such as vinblastine ( expressed as increase in michaelis constant , km ) . some neutral detergents , such as tween-20 , nonidet p-40 , and triton x-100 , were also shown to inhibit pgp atpase activity at concentrations that are required for membrane fluidization . overall , alterations in membrane structure and fluidity induced by various compounds strongly affect pgp function . therefore , it was suggested that inhibition of the transporter s activity by pluronic is at least partly due to the pluronic - induced changes in the local membrane environment ( figure 2 ) . to further understand the mechanism of pluronic sensitization of mdr cancer cells one needs to focus on the events at the subcellular level , which were characterized in great detail using p85 as an example . this copolymer exhibits evident and profound selectivity with respect to energy metabolism in mdr cancer cells . it is rapidly taken up by the cells via a caveolae - mediated endocytosis pathway and colocalizes with mitochondria already 15 min after exposure to the cells . this results in a drastic depletion of intracellular atp levels in mdr cancer cells , while non - mdr cells require significantly higher doses of pluronic to achieve similar depletion . noteworthy , the ability to deplete cellular atp levels strongly correlates with the chemosensitization properties of the copolymers in mdr cells . the selectivity of pluronic copolymers toward mdr phenotype is probably attributable to innate metabolic and physiological differences between mdr and non - mdr cells . in contrast to normal cells , that use oxidative phosphorylation for atp production , cancer cells mostly rely on glycolysis as an adaptation to hypoxic conditions in the early stages of tumor development . drug - resistant cells require more atp to support the drug efflux transporter activity and drug metabolism . adaptations leading to mdr therefore in part are associated with changes in energy metabolism to meet new energy requirements . it was shown that human breast cancer cells with acquired resistance to dox exhibit 3-fold higher glycolysis rate than their sensitive counterparts . another study by miccadei et al . found that both respiration and glycolysis rates are increased in drug - resistant ehrlich cells , resulting in almost 50% higher atp production compared to the drug sensitive cells . it was also shown that mdr cells have significantly higher activity of the respiratory chain complexes in mitochondria where nearly 50% of atp was produced , compared to only 35% of atp produced in mitochondria of sensitive cells . moreover , it was later demonstrated that mdr cancer cells have lower mitochondrial membrane potential , use fatty acids for mitochondrial oxidation when glucose becomes limited , and have high levels of expression of uncoupling protein 2 ( ucp2 ) , which results in less efficient atp synthesis . overall , the compromised mitochondrial function in mdr cells may be the achilles heel of mdr cells that allows effective and selective inhibition of atp production in drug - resistant cells . when pluronic reaches mitochondria of mdr cells , it inhibits complexes i and iv of the respiratory chain and depletes mitochondrial membrane potential . the mechanism of pluronic inhibition of respiratory chain complexes activities is not fully understood . in mitochondria in other words pluronic may act as a prooxidant , which were shown to induce apoptosis in cancer cells . noteworthy , the effects of pluronic on pgp activity , atp levels , and cytotoxicity are reversible . pgp function is restored 1 h after the removal of pluronic . at the same time the sensitization effect of pluronic is abolished in the same time frame , while it takes about 10 h to restore atp levels . interestingly , pgp expression seems to be essential for pluronic effects on respiration and atp levels . inhibition of oxygen consumption as well as atp depletion by pluronic was observed not only in drug - selected resistant cells but also in cells stably transfected with mdr1 gene , encoding pgp . inhibition of pgp with highly specific inhibitor gf120918 abolished the pluronic - induced atp depletion , while the inhibitor itself did not affect atp levels in mdr cells . pluronic effects in mdr cancer cells exhibit remarkably simple and clear structure functional relationships . the studies of the concentration dependence of the pluronic in mdr cells effect suggested that these effects are produced mainly by the copolymer single chains as they leveled up or decreased above the cmc . hydrophilic pluronics with hlb 20 and above have little if any sensitization effect in mdr cells . using pgp expressing brain microvessel endothelial cells ( bmecs ) it was demonstrated that such pluronics do not decrease membrane microviscosity , do not inhibit pgp atpase activity , practically do not internalize in the cells , and do not induce atp depletion . of all other pluronics with hlb fewer than 20 the most active in mdr cells are the copolymers with intermediate lengths of the hydrophobic ppo block from about 30 to about 60 po units . these copolymers bind with the cell membranes , decrease membrane microviscosity , and inhibit pgp atpase activity . moreover , they internalize into cells and produce atp depleting effects . the copolymers with shorter ppo blocks , fewer than 30 po units , also internalize in cells . however , they do not decrease membrane microviscosity , do not inhibit pgp atpase , and do not deplete atp . presumably , they are not sufficiently disruptive to the membrane structures to produce all these effects . the copolymers with longer ppo blocks but they do not penetrate inside the cells and do not reach mitochondria remaining stuck in the cell membranes , presumably due to their extreme hydrophobicity . accordingly notably , it was demonstrated that both atp depletion and inhibition of pgp atpase activity are essential for the sensitization of pgp overexpressing cells . when one of these factors was excluded , the drug efflux pump remained functional in both mdr cancer and pgp - expressing bmecs . oxidative stress is a condition in which the balance between the production of reactive oxygen species ( ros ) by cells and the ability to detoxify them is impaired . if oxidative stress persists , the formed peroxides and free radicals will damage all components of the cell , including membranes , proteins , and dna . accumulation of significant damage , which a cell fails to repair , will lead to apoptosis . generally , oxidative stress is associated with increased production of ros and/or decreased ability of the cell to eliminate these species . glutathione is a major cellular antioxidant that protects the cells against ros , toxins , and drugs . it is a tripeptide that exists in reduced ( gsh ) and oxidized ( gssg ) states , and normally more than 90% of cellular glutathione is in a reduced state . an accurate ratio between gsh and gssg is important to maintain the intracellular redox state , with a decrease in gsh / gssg ratio indicative of oxidative stress . gsh is also a cofactor of glutathione s - transferase ( gst ) , the major cellular detoxifying enzyme . furthermore , several members of the mrp family of abc transporters require gsh for transport activity . pluronic was shown to deplete the gsh levels and inhibit the gst activity in several mdr cell lines . inhibition of the gsh / gst detoxifying system in turn decreases the mrp - mediated efflux . the decrease of cellular gsh is also an early sign of apoptosis induced by oxidative stress , death receptor activation , or mitochondrial apoptotic signaling . one of the major sources of ros in the cells is electron transport chain in mitochondria . in normal conditions oxygen is reduced in mitochondria by cytochrome c oxidase ( complex iv ) to produce water . however , a small amount of electrons passing through the electron transfer chain reduce oxygen to produce superoxide radical . the main superoxide radical producing complexes in mitochondria are nadh dehydrogenase ( complex i ) and cytochrome bc1 complex ( complex iii ) . it is well - known that inhibition of complex i by certain inhibitors like rotenone , piericidin a , and rolliniastatin increases the ros production . as was mentioned above , pluronic quickly reaches mitochondria and inhibits complexes i and iv in mdr cells ( figure 4 ) . moreover , it stimulates the production of ros and release of cytochrome c , which are the early signs of mitochondrial apoptotic pathway . if ros are not neutralized , they induce damage of mitochondrial membrane , proteins , and dna . this leads to permeabilization of outer mitochondrial membrane , swelling of mitochondria , and release of proapoptotic proteins , like cytochrome c , apoptosis inducing factor ( aif ) , and endonuclease g. in cytoplasm cytochrome c binds to apoptosis protease activating factor ( apaf-1 ) and forms apoptosome . the activated caspase 9 in turn activates the effector caspases , which all together contribute to the completion of apoptosis . similar to atp depletion and inhibition of respiration , pluronic induced the ros formation and cytochrome c release selectively in mdr cells , while non - mdr cells did not respond in that manner . pluronic quickly enters the cells , reaches mitochondria , and induces mitochondrial membrane depolarization ( 1 ) , inhibition of complexes i ( 2 ) and iv ( 3 ) , release of cytochrome c ( 4 ) , and atp depletion ( 5 ) . in addition to induction of ros production and cytochrome c release in mdr cells , pluronic promotes drug - induced apoptosis . treatment of mdr cells with dox / pluronic p85 formulation significantly enhanced the proapoptotic signaling compared to the drug alone and inhibited the antiapoptotic defense mechanisms in vitro . it was demonstrated that dox / pluronic treatment of tumor - bearing mice significantly increased levels of caspases 8 and 9 compared to dox alone . overall , pluronic induces early as well as late stages of proapoptotic signaling in mdr cells in vitro and in vivo . inhibition of mitochondria respiratory chain complexes is most likely the main reason for increased ros production in mdr cells after treatment with pluronic . additionally , depletion of major intrinsic cellular antioxidant gsh would increase cell sensitivity to the ros . it has been shown that drug - induced ros production may be directly linked to their cytotoxic activity and that detoxification of free radicals by gsh / gst is very important in mdr cells to facilitate drug resistance . therefore , when combined with dox , pluronic not only drastically increases the drug accumulation in the cells but also promotes the apoptosis in the mdr cells . the mechanism of development of mdr in cancer remains a highly debated subject , and most likely there is no uniform theory that will apply to all cancers . it is now widely accepted that cscs play an important role in cancer development , metastasis , and development of drug resistance . cscs comprise a small cell subpopulation within the tumor with distinct functional and phenotypical characteristics . first , cscs overexpress specific markers . however , these markers differ from cancer to cancer and to date there is no uniform marker that can be used to isolate cscs from every tumor . second , cscs have unlimited ability to divide and produce cells of all other phenotypes in the tumor . third , cscs are able to form tumors when transplanted into mice and to form so - called tumorspheres when grown in anchorage independent conditions . finally , cscs are intrinsically drug resistant : they overexpress drug efflux transporters , such as pgp and bcrp , have active antiapoptotic pathways , and spend most of their time in the g0 nondividing cell cycle state , which makes them insensitive to cytostatic drugs often used in chemotherapy . therefore , cscs can avoid classical chemotherapy and repopulate the tumor , possibly leading to mdr development . moreover , there are reports suggesting that cscs phenotype is dynamic and can be acquired by non - cscs under certain conditions . overall , successful therapy needs to be equally efficient in eliminating both bulk tumor cells and cscs . in addition to mdr chemosensitization properties , pluronics also prevent the development of mdr upon selection with cytotoxic drugs in vitro and in vivo . specifically , in one study human breast carcinoma mfc7 cells were selected with dox for drug resistance in the presence or absence of p85 at concentration below cmc ( 0.001 wt % ) . the cells cultured with dox / p85 were not able to grow at concentrations of the drug exceeding just 10 ng / ml . in contrast , cells cultured with dox alone eventually developed mdr and could tolerate up to 10,000 ng / ml dox in the culture media . further analysis has shown that cells treated with dox / p85 did not overexpress pgp and , therefore , remained sensitive to the drug . in contrast , cells exposed to dox alone exhibited significant overexpression of pgp . this developed drug resistance can be resensitized by pluronic to the initial level of the drug sensitive cells . interestingly , when the cells were selected with lower concentration of dox , they were not sensitized by pluronic , even though they displayed low levels of pgp expression and detectable levels of mdr1 mrna . functional analysis of pgp activity using accumulation of pgp substrate ( rhodamine 123 ) showed that pgp in those cells was not or nearly not functional compared to more resistant cells . even though cells selected with lower concentrations of dox were not sensitized with pluronic , they showed strong atp depletion in response to pluronic treatment . moreover , it was demonstrated that selection of cells with dox and dox / p85 resulted in very different changes in the gene expression patterns in these cells . p85 alone , however , had little if any effect on the gene expression . similar results were observed in p388 murine leukemia tumor cells selected for dox resistance with or without p85 both in vitro and in vivo . overall , this suggests that simple addition of inert polymer excipient to the drug drastically changes pharmacogenomic responses of cancer cells to this drug . however , our understanding of the mechanism behind the prevention of mdr development by pluronic and alterations in gene expression profiles is very limited . in view of csc theory a small population of tumor cells is guiding tumor progression , metastasis , and mdr development . since cscs share certain characteristics of mdr cells , such as overexpression of drug efflux transporters ( pgp , bcrp ) and altered metabolic pathways , we proposed that pluronics can sensitize cscs to chemotherapeutic drugs similar to mdr cells . in a recent study using the same p388 leukemia ascitic tumor model as before , we demonstrated that dox / pluronic combination , sp1049c , comprising mixed micelles of pluronic f127 ( ( eo)100-(po)65-(eo)100 , hlb 22 , mw 12 600 g / mol ) and l61 , effectively decreases frequency of tumor initiating cells and , as a result , suppresses tumorigenicity and tumor aggressiveness in vivo . in agreement with previous findings , sp1049c also prevented the development of mdr by inhibition of bcrp overexpression . in contrast to dox alone , sp1049c depleted the tumorigenic cd133 + and aldh+ cell subpopulations . furthermore , in vitro pretreatment of ascitic cells with sp1049c significantly reduced the in vitro colony forming potential of the cells already at 10 ng / ml dox , while dox alone had the same effect at 10 times higher concentration . as mentioned above , dox / pluronic combination drastically changes the gene expression profiles in cancer cells compared to dox or pluronic alone upon continuous exposure . in this work we have shown that dna methylation patterns also change drastically upon in vivo treatment of cancer cells with sp1049c compared to saline control , polymers , or dox alone . it is well - known that misregulation of dna methylation / demethylation plays an important role in cancer origin , progression , angiogenesis , metastasis , and mdr development . sp1049c not only induced the strongest epigenetic changes but also showed very small overlap of affected genes with other treatment groups . functional analysis of affected genes done using ingenuity pathway analysis ( ipa ) has shown that the top affected biological functions and canonical pathways affected by sp1049c treatment relate to cellular function , growth , and maintenance , as well as regulation of stem cell differentiation and pluripotency . altogether , on top of mdr sensitization , the prevention of mdr development by pluronics , depletion of tumorigenic cell subpopulations , and decrease of tumorigenicity and tumor aggressiveness offer significant advantages for the development of new formulations of approved and/or experimental therapeutics . pluronic copolymers attracted a lot of attention in drug delivery and tissue engineering applications . pluronic - based micellar formulation of dox , sp1049c , was the first in class polymeric micelle drug to advance to clinical stage and has successfully completed phase ii clinical trial in advanced esophageal cancer patients . in studies in rodent and nonrodent animal models it has been demonstrated , as well as in patients , that mtd and pharmacokinetic profiles of dox alone and sp1049c are very similar . moreover , animal studies using mdr overexpressing tumors have shown that pluronic formulations in vivo exhibit key effects observed in mechanistic studies in vitro . first , noninvasive single photon emission computed tomography ( spect ) and tumor tissue radioactivity sampling demonstrated that intravenous coadministration of pluronic p85 with a pgp substrate , tc - sestamibi , greatly increases the tumor uptake of this substrate in the mdr tumors . second , p magnetic resonance spectroscopy ( p - mrs ) in live animals and tumor tissue sampling for atp suggest that p85 and dox formulations induce pronounced atp depletion in mdr tumors . finally , these formulations were also shown to increase tumor apoptosis in vivo by terminal deoxynucleotidyl transferase dutp nick end labeling ( tunel ) assay and reverse transcription polymerase chain reaction ( rt - pcr ) for caspases 8 and 9 . in phase i clinical study of sp1049c in 26 patients , maximum tolerated dose ( mtd ) and dose - limiting toxicity ( dlt ) were determined as 70 and 90 mg / m respectively . ii study 21 patients ( 19 evaluable for response ) with metastatic or locally advanced unresectable adenocarcinoma of the esophagus and gastroesophageal junction ( gej ) were treated with 75 mg / m sp1049c every 3 weeks until disease progression or unacceptable toxicity . in this study sp1049c demonstrated prominent single agent antitumor activity ( 47% objective response rate in the evaluable population , 9 partial responders , 10 month median overall survival , and 6.6 month progression free survival ) with toxicity profile similar to that of dox at equivalent dose and administration schedule . unique biological activities of pluronics in addition to their drug solubilization properties make pluronics a very attractive platform for drug delivery . for example , in recent work chen and coauthors used mixed micelles of p105 and f127 to overcome pgp - mediated mdr to methotrexate ( mtx ) in vitro and in vivo . this system has shown relatively high drug loading and ph - dependent drug release , improved pharmacokinetics , biodistribution and antitumor activity in human lung ( a549 ) and oral epidermoid carcinoma ( kbv ) mdr xenograft tumor models , and reduced systemic toxicity ( table 1 ) . the same group has also used pluronic p105/f127 mixed micelles to deliver docetaxel ( dtx ) to taxol - resistant non - small cell lung cancer . while in drug sensitive cells the micelles had similar ic50 to taxotere , in drug - resistant a549/taxol cells they demonstrated 10-fold lower ic50 compared to taxotere control ( 0.059 g / ml vs to 0.593 g / ml ) . in in vivo a549/taxol drug - resistant tumor model dtx loaded mixed pluronic micelles showed 69.05% tumor inhibition , versus 34.43% for taxotere control ( table 1 ) . in another work shen et al . developed novel pluronic - polyethylene imine ( pei)/d--tocopheryl polyethylene glycol 1000 succinate ( tpgs ) nanoparticles to overcome paclitaxel ( ptx ) drug resistance and codeliver survivin shrna . tpgs was used to improve micelle stability and drug loading , p85 was used to form micelles and inhibit gst activity , and pei was used to bind shrna . these complex nanoparticles have shown a synergistic effect in cytotoxicity experiments in a549/t ptx resistant cells , but not in parental a549 drug sensitive cells , and displayed effective antitumor activity in vivo in mdr tumor model . furthermore , the authors have shown that gst isolated from mdr cells was 3.8 times more active than extracted from sensitive cells and that both p85 and p85pei conjugate effectively inhibited only gst of mdr cells but not of non - mdr cells . this is an important observation , since gst plays an important role in ptx metabolism and its inhibition would increase accumulation of ptx in the cells . other examples that use pluronic mdr reversal properties for overcoming mdr include poly(caprolactone)-modified pluronic p105 ( p105-cl ) ptx loaded micelles developed by wang et al . these polymers displayed atp depletion , inhibition of mitochondrial function , and membrane fluidization activities , similar to what was reported before for other pluronics . a few years earlier the same group developed folate - targeted pluronic micelles for delivery of ptx and circumvention of mdr . the authors have shown that folate conjugated pluronic p105 or l101 ptx loaded micelles better accumulate in mcf7/adr cells and have significantly higher efficiency compared to nontargeted micelles of ptx alone ( table 1 ) . the biological response - modifying properties are , however , not unique to pluronics . a number of other natural and synthetic polymers have been reported to inhibit drug efflux transporters . for example , polymers developed by cambon and colleagues with similar architecture to pluronics , but with poly(styrene oxide ) ( pso ) instead of ppo , also form micelles which have shown efficient drug loading and ph - dependent release , as well as pgp inhibition activity . furthermore , in another study from the same group the authors evaluated the structure activity relationships of nearly 30 copolymers with structures similar to pluronics , but containing different hydrophobic blocks , including propylene oxide , lactide , methylene , butylene oxide , valerolactone , caprolactone , styrene oxide , and glycidyl . many of the screened copolymers induced increase of dox accumulation in the pgp overexpressing mdr cells , as well as inhibition of pgp atpase activity . notably , the most active copolymers had longer hydrophobic chains compared to what is considered optimal for pluronics , that is , pluronics with intermediate length of hydrophobic block and relatively low hlb . furthermore , tpgs was also reported to inhibit pgp . tpgs is a common form of vitamin e , and it has been recognized as a potent enhancer of oral absorption of drugs due to inhibition of drug efflux transporters . compared tpgs with different peg lengths ( 2006000 ) and have found that commercial tpgs-1000 is one the most potent analogues in the series of polymers . other pharmaceutical excipients , including some tweens ( pegylated sorbitanes ) , brij ( alkyl - peo surfactants ) , and myrj ( peo - stearates ) , also demonstrated pgp inhibition , that strongly depends on hlb of the polymer , albeit they generally remain less potent than pluronics . altogether , there are number of polymers that possess the advantageous properties of inhibition of drug efflux transporters and can be used to overcome cancer mdr or to improve oral drug bioavailability . pluronics , however , represent the most studied group of potent polymers with respect to molecular mechanism of pgp inhibition and mdr sensitization . considering similar activities observed in other groups of polymers , it is likely that some general patterns of structure activity relationships of pluronics ( hlb , architecture , etc . ) and spectrum of biological effects can be extrapolated to other amphiphilic polymers . intrinsic and acquired drug resistance represents the great obstacle for successful treatment of cancer . numerous approaches have been utilized in attempts to overcome drug resistance with limited success . in this review we have discussed the biological properties of pluronic block copolymers and other polymers with similar biological activities , which , in addition to carrier function , make them an attractive platform for drug delivery . the mdr chemosensitization activity of pluronics ( and other surfactants ) has been known for a while now , and the mechanisms have been extensively studied ( figure 5 ) . however , we are still far from complete understanding of how exactly pluronics interact with mdr cells and why these effects are specific to mdr phenotype . recent studies have shown that combination of chemotherapeutic drug ( dox ) with pluronic effectively depletes tumorigenic cell subpopulation and decreases tumorigenicity and tumor aggressiveness . this finding being so simple by nature drastically changes the whole concept from pluronics being just another mdr modulator to a class of agents that might help to combat cancer at its root by killing cscs . on the other hand , we now have even more questions regarding the mechanism of action of pluronic than we had before . we believe that thorough understanding of these mechanisms will allow better design of pluronic ( and similar polymers)-based drug delivery systems for effective cancer therapy . ( a ) pluronic binding with plasma membrane of mdr cancer cells ( 1 ) induces membrane fluidization , disruption of membrane microdomains , and inhibition of drug efflux transporters activity ( pgp shown as an example ) . pluronic also reaches mitochondria where it ( 2 , 3 ) inhibits complexes i and iv of mitochondria respiratory chain and ( 3 ) induces inner mitochondrial membrane depolarization . this ( 4 ) results in atp depletion and ( 5 ) promotes cytochrome c release and ros generation in mdr cells . altogether , the mdr cells respond to a dox / pluronic combination by ( 6 ) an increased proapoptotic signaling and decreased antiapoptotic defense . ( b ) moreover , dox / pluronic combination effectively depletes tumorigenic subpopulation of cscs , prevents development of mdr , and significantly alters dna methylation and gene expression profiles .
multidrug resistance ( mdr ) remains one of the biggest obstacles for effective cancer therapy . currently there are only few methods that are available clinically that are used to bypass mdr with very limited success . in this review we describe how mdr can be overcome by a simple yet effective approach of using amphiphilic block copolymers . triblock copolymers of poly(ethylene oxide ) ( peo ) and poly(propylene oxide ) ( ppo ) , arranged in a triblock structure peo - ppo - peo , pluronics or poloxamers , raised a considerable interest in the drug delivery field . previous studies demonstrated that pluronics sensitize mdr cancer cells resulting in increased cytotoxic activity of dox , paclitaxel , and other drugs by 23 orders of magnitude . pluronics can also prevent the development of mdr in vitro and in vivo . additionally , promising results of clinical studies of dox / pluronic formulation reinforced the need to ascertain a thorough understanding of pluronic effects in tumors . these effects are extremely comprehensive and appear on the level of plasma membranes , mitochondria , and regulation of gene expression selectively in mdr cancer cells . moreover , it has been demonstrated recently that pluronics can effectively deplete tumorigenic intrinsically drug - resistant cancer stem cells ( csc ) . interestingly , sensitization of mdr and inhibition of drug efflux transporters is not specific or selective to pluronics . other amphiphilic polymers have shown similar activities in various experimental models . this review summarizes recent advances of understanding the pluronic effects in sensitization and prevention of mdr .
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Proceed to summarize the following text: senescence was first described as a state of irreversible growth arrest that normal human fibroblasts enter at the end of their replicative lifespan . this phenomenon has been observed in a variety of somatic cells derived from many species , which is in contrast to the infinite replicative capacity displayed by germline , cancer , and certain stem cells . senescent cells are irreversibly arrested in g1/g0 phase of the cell cycle and lose the ability to respond to growth factors [ 3 , 4 ] . they show sustained metabolic activity for long periods of time and become resistant to apoptosis [ 6 , 7 ] . in addition , senescent cells undergo distinctive changes in morphology to a flat and enlarged cell shape and are often accompanied by the induction of acidic senescence - associated -galactosidase ( sa--gal ) activity . at the molecular level , alterations in gene expression specific to senescent cells have been identified [ 1014 ] , including those constituting senescence - associated secretome , which can trigger profound changes in the surrounding cells and microenvironment [ 1517 ] . the changes of gene expression in senescent cells can be partially explained by alterations in chromatin structure , including the formation of senescence - associated heterochromatic foci ( sahf ) , which is associated with trimethylated lysine 9 of histone h3 , heterochromatin protein 1 , and high - mobility group a protein [ 1820 ] . the formation of sahf requires the recruitment of prb to e2f - responsive promoters and is responsible for the stable repression of e2f target genes , possibly contributing to the irreversibility of senescence . the onset of replicative senescence is determined by the number of times that a population of cells can divide , suggesting that a mitotic clock recording cell divisions governs this cellular process [ 21 , 22 ] . the discovery that telomeres get progressively shortened with each cell division provides a plausible explanation for the nature of this mitotic clock [ 2326 ] . because of the inability of dna polymerases to replicate dna at the very ends of linear chromosomes , telomeres become progressively shortened during successive cell divisions [ 23 , 27 , 28 ] . telomerase , which is responsible for de novo synthesis of telomeric repeats and maintenance of telomere length , is expressed in germline , stem and cancer cells , but undetectable in majority of normal somatic cells [ 30 , 31 ] . in the absence of telomerase , progressive telomere shortening is thought to be the major cause of replicative senescence . supporting this notion , enforced expression of the telomerase catalytic subunit ( tert ) has been shown to prevent telomere shortening and extend the lifespan of human somatic cells [ 3234 ] . conversely , inhibition of telomerase in immortal cells has been found to limit their replicative lifespan [ 35 , 36 ] . critically shortened telomeres lose the protection of telomere - binding proteins , leading to telomere uncapping . recent studies have revealed that dna damage foci containing multiple dna damage - response proteins , such as 53bp1 , h2ax , mdc1 and mre11 , are found at telomeres in senescent cells , suggesting that uncapped telomeres are recognized as dna breaks and thus trigger a dna damage response [ 3739 ] . in addition to telomere attrition , senescence can be activated by many types of stress , including aberrant activation of certain oncogenes [ 4042 ] , damage to chromatin structure [ 4345 ] , oxidative stress [ 4648 ] , dna damage [ 49 , 50 ] , and inadequate culture conditions [ 48 , 51 , 52 ] . collectively , they are referred to as stress - induced premature senescence . among these senescence - inducing stimuli , oxidative stress has been shown to accelerate telomere shortening [ 47 , 53 ] , possibly by inducing telomeric single - strand breaks . however , stress - induced premature senescence , unlike replicative senescence , is largely independent of the telomere length or the number of cell divisions [ 5557 ] . the final outcome of both replicative senescence and stress - induced premature senescence is remarkably similar in that they share common changes in cell cycle regulation and morphological properties [ 40 , 41 , 46 , 49 , 50 , 58 , 59 ] . although gene expression pattern can vary depending on the specific types of tissues and cells or the specific stimuli to trigger the senescence response , senescent cells display a unique pattern of gene expression that differs from proliferating cells or quiescent cells . in addition to the cell cycle regulatory genes , the expression of dna damage checkpoint genes , inflammation and stress - associated genes , genes encoding extracellular matrix proteins and extracellular matrix - degrading enzymes , and cytoskeletal genes and metabolic genes is generally altered during replicative and premature senescence . recent studies suggest that dna damage could be a common cause for different forms of senescence induced by various stimuli [ 11 , 12 , 14 , 6063 ] . senescence is now considered as a general stress response in normal cells to various types of cellular damage . despite the commonality shared by senescence induced by various stimuli , regulation of senescence varies significantly among cells derived from different species , or even different types of cells from the same species . for example , telomere shortening is the major cause of senescence in human fibroblasts , whereas mouse fibroblasts undergo senescence that is independent of telomere shortening and probably mediated by oxidative stress [ 48 , 52 ] . however , these pathways seem to converge on p53 and prb , and inactivation of both the p53 and prb pathways is often required to prevent the activation of senescence [ 6670 ] . in senescent cells , p53 is phosphorylated and its transactivation activity is elevated , although its mrna and protein levels are largely unchanged [ 38 , 7174 ] . dna damage response elicited by telomere dysfunction leads to activation of atm / atr and chk1/chk2 , which in turn phosphorylate and stabilize p53 [ 3739 , 75 , 76 ] . in addition , p53 is activated and plays an important role in stress - induced premature senescence [ 40 , 50 , 7779 ] . this p53 activation is mediated by p14 ( or p19 in mouse ) encoded by the ink4a / arf locus . arf stabilizes p53 by sequestering mdm2 , an e3 ubiquitin ligase targeting p53 for degradation . inactivation of p53 or arf in mouse embryo fibroblasts ( mefs ) is sufficient to prevent senescence [ 8183 ] . one of the p53 targets is p21 ( p21 ) , whose increased expression transactivated by p53 is responsible for cell cycle arrest . this p21 up - regulation is dependent on signal(s ) initiated by telomere shortening , as expression of tert blocks this up - regulation [ 8991 ] . overexpression of p21 is able to induce a senescence - like growth arrest in some cells [ 92 , 93 ] , while deletion of p21 can postpone senescent arrest [ 94 , 95 ] . collectively , these studies suggest that p53 regulates senescence at least in part by inducing p21 . as a cyclin - dependent kinase inhibitor , up - regulation of p21 in senescent cells leads to inhibition of prb phosphorylation , which controls cell cycle progression [ 18 , 84 ] . there are instances that inactivation of either p53 or prb can significantly delay the onset of senescence , supporting a linear p53-prb pathway [ 68 , 96 ] . in many other instances , both p53 and prb need to be inhibited to prevent replicative senescence , suggesting two independent pathways [ 6669 ] . in parallel to p21 , p16 ( p16 ) the expression of p16 is increased during replicative senescence [ 88 , 9799 ] , but whether increased p16 expression is regulated by telomere shortening is controversial . as telomere shortening is the major cause of replicative senescence in human fibroblasts , and inactivation of both the p53 and prb pathways is required to prevent replicative senescence , it is reasonable to expect that dysfunctional telomeres may signal into p16-prb axis . . the increased expression of p16 occurs after senescence has already been established in culture [ 88 , 97 , 98 , 100 ] , in contrast to the rapid increase of p21 expression in cells approaching replicative senescence . within a senescent population of human cells , some cells express p16 , while others express p21 [ 38 , 96 , 100 ] . dna damage foci at telomeres are found only in cells expressing p21 , but not in p16 positive cells , suggesting that p16 elevation is independent of telomere shortening . consistent with this notion , p16 induction during senescence , unlike p21 , is not prevented by ectopic expression of tert . the expression of p16 is readily increased during premature senescence induced by a variety of stress [ 4042 , 49 , 51 , 101 ] . it is not entirely clear how p16 expression is regulated by various senescence signals [ 102104 ] . under certain circumstances , p16 is coordinately regulated with arf , which is also encoded by the ink4a / arf locus . for example , polycomb complex proteins have been shown to repress the ink4a / arf locus [ 100 , 105108 ] . decreased expression of polycomb complex proteins relieves the repression of the ink4a / arf locus and is responsible , at least in part , for the elevation of p16 and arf in senescent cells [ 100 , 106 , 107 ] . id1 , whose expression is decreased in senescent cells , has been shown to specifically suppress p16 expression by forming heterodimers with transcriptional factors ets1/2 or e47 and inhibiting their ability to transactivate p16 [ 110112 ] . down - regulation of id1 in human and mouse fibroblasts has been shown to induce p16 expression and senescence [ 110 , 112 ] , while ectopic expression of id1 delays senescence in human fibroblasts , mammary epithelial cells , keratinocytes , and endothelial cells [ 98 , 113115 ] , suggesting an important role for id1 in regulating p16 and senescence . the expression of p16 varies significantly among different human cell lines , and this variable expression seems to hold the key to as whether p53 and prb function in a linear manner or in parallel . in cells with low or no p16 expression , p53 and prb may function in a linear pathway , whereas p53 and prb work in parallel in cells with significant p16 expression . in mouse embryo fibroblasts ( mefs ) , inactivation of p53 or arf , but not p16 , is sufficient to prevent senescence [ 8183 , 116 ] , indicating that p53-arf axis is the major regulator of senescence pathway in mouse cells . human mammary epithelial cells quickly encounter a growth arrest state that is not associated with telomere shortening but mediated by p16 up - regulation [ 33 , 101 ] . a subset of cells with p16 inactivation emerge from the arrest population and continue to divide until reaching a second growth arrest state that is associated with telomere shortening [ 33 , 51 , 101 ] . depending on cell types , culture conditions , and the extent of stress , inactivation of either p53-p21-prb or p16-prb pathway individually , or both pathways together , is required to prevent senescence . tumorigenesis is a multistep process , in which a normal cell acquires mutations in a number of cancer - causing genes . by restricting cell proliferation and thereby impeding the accumulation of mutations , senescence acts as an important tumor suppression mechanism . furthermore , senescence induced by aberrant activation of oncogenes , oxidative stress , or dna damage prevents cells at risk of malignant transformation from proliferating [ 55 , 59 , 118 , 119 ] . senescence represents a physiologic response that cells must overcome in order to divide indefinitely and develop into tumors . consistent with the notion that senescence is a tumor suppression mechanism , well - established tumor suppressors , including p53 , prb , p16 , arf , and p21 , are regulators of senescence [ 102 , 118 , 120 ] . in contrast to normal somatic cells , cells derived from tumors divide indefinitely in culture . the ability to escape senescence ( i.e. , immortality ) is a necessary step for cells to become transformed and one of the hallmarks of cancer cells . 80%90% of human cancer cells acquire unlimited proliferative potential through reactivation of telomerase [ 30 , 31 ] , while the rest maintain telomere length by a recombination - mediated process termed alternative lengthening of telomeres [ 121 , 122 ] . these observations in human cancer strongly suggest a connection between telomere checkpoint and tumor suppression . supporting this connection , inhibition of telomerase activity in cancer cells limits their growth by triggering telomere shortening and cell death [ 35 , 36 ] . conversely , ectopic expression of telomerase in normal human cells leads to immortalization and enhances the ability of these cells to be neoplastically transformed [ 33 , 34 , 123 ] . genetic studies in mice deficient in telomerase provide further support for telomere shortening as a tumor suppression mechanism . mice deficient in the telomerase rna component ( mterc ) gradually lose telomeres over several generations , and tumorigenesis is significantly reduced in late generations of mterc mice with telomere attrition [ 130140 ] . decreased tumorigenesis is also observed in late generation of mice with a null mutation in telomere catalytic subunit ( mtert ) , and p53 mutation enables tumor progression in these mice . more importantly , two recent studies provide evidence that senescence induced by telomere shortening is responsible for tumor suppression [ 142 , 143 ] . when apoptosis is blocked by the expression of bcl-2 or a specific p53 mutant ( r172p ) , shortened telomeres reduce tumorigenesis in mterc mice . suppression of tumor development requires p53-dependent activation of senescence [ 142 , 143 ] , demonstrating that senescence induced by telomere shortening is an effective tumor suppression mechanism in vivo . the discovery that oncogenic ras protein can induce a senescent arrest after causing an initial hyperproliferation in normal cells suggests that induction of senescence is an intrinsic cellular response that prevents cells at risk from proliferating . in mouse tumor models with oncogenic ras , senescent cells are found in premalignant lesions in lung , spleen , breast , and pancreas . the observation of senescent cells has been extended to many premalignant lesions or benign tissues induced by different oncogene activation or tumor suppressor inactivation in mouse [ 147155 ] and human [ 148 , 156159 ] . importantly , senescent cells are absent in malignant tumors [ 61 , 144 , 145 , 147150 , 152 , 156 , 158 , 160 ] , suggesting that oncogene - induced senescence is a powerful tumor suppression mechanism by restricting proliferation of cells with oncogenic mutations and this senescence block must be evaded for malignancy to progress . consistently , deletion of senescence regulators such as p53 , arf , p16 , p27 , suv39h1 or prak abrogates senescence and causes progression of tumors to the malignant stage [ 144146 , 148150 , 152 , 153 , 160 ] . in theory , senescence offers an attractive therapeutic option if it can be induced in tumor cells . because of the uncertainty in reactivating in cells , a response that otherwise has been overcome , senescence remains as an underappreciated therapeutic approach [ 161 , 162 ] . surprisingly , many cancer cells retain the ability to senesce either spontaneously or in response to external stress stimuli , even though most cancer cells have overcome the senescent arrest during tumorigenesis . as tumors often develop resistance to apoptosis induced by anticancer treatment , induction of senescence in tumor cells serves as an alternative approach in cancer therapy , and could be especially effective in treatment of cancer cells in which apoptotic pathways are disabled . as telomerase is critical for the maintenance of telomere length , inhibition of telomerase in cancer cells leads to shortening of telomeres , which is a major cause of senescence activation [ 24 , 33 , 34 ] . since 8090% of human cancers acquire unlimited proliferative potential through activation of telomerase [ 30 , 31 ] , the strategy of inhibiting telomerase in cancer therapeutics targets a broad range of malignancies . in addition , this approach offers desired specificity in targeting cancer cells , as telomerase is expressed in most cancer cells , but undetectable in the majority of normal cells including adult stem cells [ 164 , 165 ] . the emerging cancer therapeutics targeting telomerase include small molecule or oligonucleotide inhibitors of telomerase enzymatic activity , antitelomerase immunotherapy , inhibitors of telomerase expression and telomere - disrupting agents [ 166168 ] . although apoptosis is induced by inhibition of telomerase in some studies , induction of senescence as a result of telomerase inhibition is clearly demonstrated to be responsible for tumor suppression [ 169173 ] . the effectiveness of these approaches has been demonstrated in many studies [ 174177 ] , and several clinical trials targeting telomerase for cancer therapeutics are now ongoing . senescence induced by oncogene activation or inactivation of tumor suppressor genes must be evaded for tumors to progress to full malignancy , which is often associated with inhibition of crucial senescence regulators . reactivation of senescence response offers a great opportunity in cancer therapeutics . considering the critical role of p53 in senescence regulation and common occurrence of p53 mutations in cancer cells various approaches have been developed to target p53 in order to restore normal p53 function in cancer cells , including pharmacological depletion of mutant p53 protein [ 178 , 179 ] , restoring normal function in mutant p53 [ 180 , 181 ] , and reactivation of p53 [ 182189 ] . in most of these reports , senescence as a tumor suppression mechanism after restoring p53 expression has been demonstrated in two recent elegant studies [ 190 , 191 ] . in a mouse model of hepatocellular carcinoma , reactivation of p53 in these tumors results in rapid activation of senescence and subsequent immune cell infiltration which leads to clearance of tumor cells . in a separate study , restoration of p53 in p53-deficient mouse models of lymphoma , and osteosarcoma leads to tumor regression . apoptosis is selectively induced by p53 in lymphomas , while senescence induced by p53 in osteosarcomas is responsible for tumor regression , suggesting that tissue type and/or genetic context play a critical role in determining the cellular response in p53-mediated tumor regression . taken together , restoration of p53 function in tumors offers an effective way to restrict tumor growth by inducing senescence or apoptosis . as p16 and p21 have been shown to induce senescence efficiently , these senescence regulators together with arf and prb may provide additional targets for the effective activation of senescence in cancer therapeutics . in addition to restoration of tumor suppressor genes , oncogene inactivation offers another possible intervention to induce senescence in cancer cells . suppression of c - myc oncogene induces senescence and leads to tumor regression in diverse tumor types including hepatocellular carcinoma , lymphoma and osteosarcoma . senescence induced by myc inhibition depends on critical senescence regulators such as p53 , p16 or prb.inactivation of these senescence regulators impairs senescence induction and tumor regression . inhibition of myc as therapeutic intervention is further illustrated in lung carcinoma mouse model initiated by oncogenic ras . these studies indicate that senescence response not only is functional in cancer cells , but also can be reactivated to cause tumor regression . furthermore , these studies suggest that therapeutic intervention aimed at molecules required to support tumor growth may also lead to senescence induction and ultimately tumor regression . the finding that senescence can be induced by dna damage [ 49 , 50 ] suggests that chemotherapeutic drugs , which cause dna damage , may activate senescence in tumor cells and therefore determine the drug response in cancer treatment . chemotherapeutic drugs induce senescence in various types of tumor cells in culture [ 195199 ] . in a myc - driven mouse lymphoma model , chemotherapeutic drug cyclophosphamide induces p53- and p16-dependent senescence in lymphomas , leading to better prognosis following chemotherapy . in human breast cancer , a high percentage of tumors after chemotherapy show positive staining for senescence markers , and induction of senescence in these tumors is associated with p53 and p16 . induction of senescence is not observed in tumors before chemotherapy , suggesting that senescence observed in tumors is induced by chemotherapy . taken together , these studies show that senescence induction can positively influence the outcome of cancer treatment . senescence - inducing drugs may be effective alone or in combination with classic therapeutic approaches to reduce tumor growth and toxicity to normal cells . aging is characterized by progressive deterioration of physiological function in all tissues and organs after a period of development . this biological process is associated with increased susceptibility to major chronic diseases and ultimately mortality . since the discovery of senescence in cultured cells , it is recognized that cellular senescence and organismal aging may be closely related because of their shared ability to limit lifespan . it is hypothesized that constant tissue regeneration results in accumulation of senescent cells in somatic tissues , which limits tissue renewal , perturbs normal tissue homeostasis and ultimately leads to aging [ 59 , 118 , 200 ] . cells with characteristics of senescence have been reported to increase with advancing age in mice , primates and humans [ 9 , 201206 ] . in addition , accumulation of senescent cells is linked with age - associated pathological conditions , such as osteoarthritis , atherosclerosis [ 208211 ] , dementia , liver cirrhosis , and respiratory disease [ 213 , 214 ] . the initial support for the senescence theory of aging comes from the observation of an inverse correlation between the in vitro lifespan of cells and the age of donors from which they are derived [ 215219 ] , although this correlation has been disputed . subsequent support comes from studies of cells derived from progeroid patients , such as werner syndrome , which achieve fewer cell divisions before entering senescence than cells derived from normal individuals of same age . direct evidence supporting senescence as one of the aging mechanisms , however , is still missing . it remains to be determined whether accumulation of senescent cells is responsible for aging or age - related diseases . it is evident that telomere shortening occurs in aged human tissues [ 222235 ] , at sites of age - related pathological conditions [ 203 , 236243 ] , or associated with stress and obesity [ 244 , 245 ] . although it remains to be demonstrated whether telomere shortening leads to the accumulation of senescent cells in vivo , and more importantly makes a substantial contribution to aging , studies of human premature aging syndromes support a link between telomere attrition and aging . patients of dyskeratosis congenita and aplastic anemia have mutations in telomerase rna or catalytic subunit [ 246248 ] , and are characterized by accelerated telomere shortening [ 239 , 246 ] . further evidence for a role of telomere attrition in aging comes from genetic studies of mice deficient in telomerase . while mice with a null mutation in telomerase rna ( mterc ) are apparently normal in early generations , these mice in later generations gradually lose telomeres and show accelerated aging phenotypes [ 140 , 249 ] . similarly , premature aging phenotypes are observed in mterc mice on a cast / eij background , which have shorter and more homogenous telomere length than c576bl/6 strain . even with the presence of telomerase , shortened telomeres in mterc mice on cast / eij background are associated with premature aging . a recent study shows that telomerase reactivation can reverse much of the premature aging phenotypes in telomerase - deficient mice , indicating that telomere attrition plays a critical role in aging . furthermore , mutations in wrn or blm in the telomere dysfunctional background in mouse cause premature aging phenotypes that are characteristics of werner or bloom syndrome in human . such premature aging phenotypes are absent in mice with wrn or blm mutation but with long telomeres [ 252 , 253 ] . these studies clearly establish a link between telomere attrition and aging . whether this link is mediated through senescence triggered by telomere shortening premature aging phenotypes in late generation mterc mice are associated with reduced renewal capacity in highly regenerative tissues such as skin , intestine , bone marrow and reproductive organs [ 140 , 249251 ] , suggesting that stem cells may be affected by telomere shortening . tissue - specific or adult stem cells , which are capable of self - renewal and differentiation , are essential for the normal homeostatic maintenance and regenerative repair of tissues throughout the lifetime of an organism . the self - renewal ability of stem cells is known to decline with advancing age , eventually leading to the accumulation of unrepaired , damaged tissues in old organisms [ 59 , 254256 ] . by limiting cell proliferation , senescence in stem cells is hypothesized to contribute to aging by reducing the renewal capacity of these cells [ 21 , 59 , 118 ] . for example , human mesenchymal stem cells have no detectable telomerase activity , and hematopoietic stem cells from human and mouse have low level of telomerase activity [ 258260 ] . it is possible that senescence induced by telomere attrition may occur in stem cells over the lifespan of an organism and would result in the reduction of the renewal capacity of stem cells . however , it remains to be determined whether stem cells undergo senescence during aging . the expression of p16 increases with advancing age in humans and rodents [ 264270 ] . increase of p16 in aged rodents is attenuated in several tissues ( adrenal , heart , kidney , ovary , and testis ) by caloric restriction , which potently slows aging . moreover , age - related increase of p16 is found to be associated with a decline in the renewal capacity of stem cells in brain , pancreas , and hematopoietic system , and these stem cells derived from aged mice lacking p16 have increased regenerative potential [ 271273 ] . it has been shown that p21 is required to maintain quiescence of hematopoietic stem cells ( hscs ) . in the absence of p21 , increased cell cycling leads to stem cell exhaustion , which is responsible for impaired self - renewal of hscs . interestingly , deletion of p21 in late generation mterc mice improves stem cells function and rescues much of the premature aging phenotypes associated with telomere attrition . hscs from p53-deficient mice have increased stem cell population and enhanced renewal capacity [ 276 , 277 ] . furthermore , mice with excessive p53 activity maintain cancer protection , but age prematurely including impairment of hscs [ 279282 ] , which is at least in part due to increased sensitivity to senescence - inducing stimuli . interestingly , concomitant increase of normal p53 and p19 leads to increased longevity in mice , although elevation of p53 alone is not sufficient to increase longevity [ 284 , 285 ] . collectively , these recent studies support an emerging link between senescence regulation and aging , and show the potential importance of senescence regulation in stem cells aging . senescence is regarded as an antagonistic pleiotropy : beneficial as a tumor suppressor , but detrimental to organisms by contributing to aging . great progress has been made in our understanding of senescence regulation in cancer and aging . the exact function of senescence - associated secretome in cancer and aging is of great interest and needs to be investigated . investigation of telomere shortening and senescence in stem cells during physiological aging is much needed for our understanding of the role of senescence in aging , which leads to the intriguing question as whether inhibition of senescence may slow aging .
senescence is regarded as a physiological response of cells to stress , including telomere dysfunction , aberrant oncogenic activation , dna damage , and oxidative stress . this stress response has an antagonistically pleiotropic effect to organisms : beneficial as a tumor suppressor , but detrimental by contributing to aging . the emergence of senescence as an effective tumor suppression mechanism is highlighted by recent demonstration that senescence prevents proliferation of cells at risk of neoplastic transformation . consequently , induction of senescence is recognized as a potential treatment of cancer . substantial evidence also suggests that senescence plays an important role in aging , particularly in aging of stem cells . in this paper , we will discuss the molecular regulation of senescence its role in cancer and aging . the potential utility of senescence in cancer therapeutics will also be discussed .
You are an expert at summarizing long articles. Proceed to summarize the following text: recently , it was reported that exercises using whole - body vibration ( wbv ) machines have been attracting much attention from athletes and the general public . these exercises generate positive effects , as they can improve muscle function in older adults and individuals with functional problems1,2,3,4 . wbv exercises are advantageous as they pose a lower risk of injury and can improve muscle strength in shorter periods of time when compared to other resistance exercises , particularly as they can be performed without an external load5 . as the use of wbv exercises increase , a number of studies have been conducted to investigate the activation of lower extremities caused by the change in vibration intensity , as well as to propose optimized vibration intensities in a short period of time . when comparing the training intensities of the wbv exercises and conventional squat motions that target male and female college students , both of these two exercise methods offered gradual improvement in the vibration intensity and muscle activations of the lower extremities and lumbar region , and they both showed similar lower extremity and lumbar muscle activation4 . when investigating the optimized representative muscle activations of the upper / lower extremities with the wbv exercise that targeted male college students , muscle activations in the upper / lower extremities were increased at higher frequencies ( 35 hz , 40 hz , and 45 hz ) and at higher amplitudes ( 4 mm)6 . in the case of female volleyball players , the activation of the lower vastus lateralis muscle according to vibration frequency ( isometric , 30 hz , 40 hz , and 50 hz ) in the wbv exercise was higher than that in the no - vibration exercise . the greatest muscle activation was shown at 30 hz7 . in a study on the lower extremity muscle power to investigate the optimized frequency , amplitude , and duration for short - term wbv exercises that target healthy adult males and females , the correlation between vibration stimulation and time was not disclosed ; however , high frequencies and high amplitudes , as well as low frequencies and low amplitudes , were effectively related8 . as was shown in the aforementioned studies , it was generally found changes in the vibration intensity of wbv exercises affect the activation of various muscles . however , a general consensus about the optimized vibration intensity has not yet been derived . in addition , further multidirectional studies on the effects of wbv exercises on muscles in the entire body , as well as lower - extremity muscles , need to be performed , and current studies on muscle activation in each muscle are also insufficient . therefore , this study will investigate the effects of wbv stimulation on the activation of representative muscles in the human body , particularly in response to various intensities using different amplitudes and frequencies , as well as effective vibration intensity . the subjects in this study included 10 healthy men in their 20s without orthopedic disease ( age : 22.5 1.7 years ; height : 180.8 7.5 cm ; weight : 79.4 9.2 kg ) . the subjects received a complete explanation of the objective and contents of this study , as well as the study procedures , subjects rights , and safety issues . following that , they voluntarily agreed to participate in the study , and signed an informed consent form in accordance with the ethical standards of the declaration of helsinki . the subjects stood on the vibration exercise machines ( tt2590p ; turbosonic , seoul , korea ) in comfortable positions with dangling arms . they were exposed to either no - vibration or each vibration stimulus for 10 seconds ; they stood with their feet shoulder - width apart while staring straight ahead . they had 5-minute break times after each intensity to minimize the effect caused by each stimulus . the analysis intensity was set at 10 hz , 25 hz , and 40 hz for a volume of 50 , and 10 , 25 , and 40 hz for a volume of 80 ; the measurement sequence was selected at random . a bagnoli 8-channel wireless electromyography system ( delsys , boston , ma , usa ) was used to measure the muscle activation . the electrodes were attached to 8 muscle bellies : the lateral gastrocnemius , soleus , tibialis anterior , vastus lateralis , and biceps femoris in the lower extremities , and the rectus abdominis , erector spinae , and trapezius upper fiber in the body . to minimize the skin resistance , the electrode areas were shaved and cleaned with cotton soaked in alcohol to increase the adhesive property of the electrodes and to minimize any effects on the measurement . the cutoff frequency of the emg signal was set to 20450 hz and the common mode rejection ratio ( cmrr ) of the electrodes was set to 110 db . for the emg data , the intermediate 5-second section was analyzed excluding the front / rear 2.5-second sections from the total exposure time of 10 seconds . to compare the relative increases / reductions , a standing posture with no vibration stimulation was set as 100% , and the measured data for each intensity were normalized , compared , and analyzed . the collected data were statistically analyzed using the spss software for windows , version 18.0 ( ibm corporation , armonk , ny , usa ) . the mean ( m ) and the standard deviation ( sd ) for the results of each factor were calculated , and repeated - measures anova with bonfferoni correction were performed to compare the differences by the intensities in each muscle . with respect to the results of the measurement , the muscle activation effects were different in all 8 muscles according to the intensities of the wbv stimulation ; these differences were statistically meaningful ( p<0.01 and p<0.001 ) . the volume tended to have greater muscle activation at 80 than at 50 . the frequency ( hz ) tended to be maximized at 10 hz . the maximum emgrms values were found at 10 hz in 80 volume of sol , vl , bf , ra , and es muscles , and at 25 hz in 80 volume of lg , ta , and tra muscles ( table 1table 1.muscle activation of the different whole - body vibration intensity ( unit : v)vol.hzlgsoltavlbfraestrano vibration0.12 0.020.27 0.070.16 0.060.14 0.040.19 0.040.14 0.040.15 0.030.10 0.0150100.20 0.060.33 0.090.17 0.060.14 0.060.30 0.150.19 0.040.17 0.050.14 0.04250.18 0.030.35 0.070.20 0.090.24 0.100.26 0.090.17 0.040.17 0.030.14 0.02400.14 0.020.37 0.090.13 0.040.15 0.040.15 0.020.12 0.020.15 0.030.11 0.0280100.21 0.070.45 0.120.19 0.070.28 0.110.36 0.140.22 0.070.21 0.050.16 0.04250.25 0.070.42 0.100.26 0.120.24 0.080.35 0.100.20 0.050.20 0.030.18 0.06400.16 0.030.37 0.070.17 0.060.16 0.060.23 0.050.14 0.030.16 0.030.13 0.02significance**********************lg : lateral gastrocnemius ; sol : soleus ; ta : tibialis anterior ; vl : vastus lateralis ; bf : biceps femoris ; ra : rectus abdominis ; es : erector spinae ; tra : trapezius upper fiber . * in addition , the maximum emgrms values were 2.16 times greater in lg , 1.65 times greater in sol , 1.59 times greater in ta , 1.94 times greater in vl , 1.92 times greater in bf , 1.55 times greater in ra , 1.39 times greater in es , and 1.72 times greater in tra than the values in the no - vibration condition ( table 2table 2.normalized muscle activation of the different whole - body vibration intensity ( unit : % ) vol.hzlgsoltavlbfraestrano vibration1111111150101.751.221.0211.611.331.121.31251.541.301.221.641.421.171.121.33401.231.360.821.010.810.8411.0580101.781.651.141.941.921.551.391.51252.161.561.591.691.901.421.301.72401.371.351.061.091.230.951.091.28lg : lateral gastrocnemius ; sol : soleus ; ta : tibialis anterior ; vl : vastus lateralis ; bf : biceps femoris ; ra : rectus abdominis ; es : erector spinae ; tra : trapezius upper fiber ) . lg : lateral gastrocnemius ; sol : soleus ; ta : tibialis anterior ; vl : vastus lateralis ; bf : biceps femoris ; ra : rectus abdominis ; es : erector spinae ; tra : trapezius upper fiber . * * p<0.01 ; * * * p<0.001 lg : lateral gastrocnemius ; sol : soleus ; ta : tibialis anterior ; vl : vastus lateralis ; bf : biceps femoris ; ra : rectus abdominis ; es : erector spinae ; tra : trapezius upper fiber wbv exercises are known to improve not only muscular functions , but also cardiovascular functions , particularly given its relatively short exposure , which safely and comfortably stimulates the muscles , skeletal system , and circulatory system . the applicability of wbv exercises has widely expanded , especially in light of the diverse nature of related studies that have been carried out9,10,11 . the intensity of wbv is controlled by its amplitude and frequency ; however , the current method used to optimize the exercise intensity setting , which is currently based according to the purpose of each exercise , is not sufficient . in this study , muscle activation was analyzed to determine whether the wbv can improve muscular functions while participants were in a standing position . muscle activity was measured after selecting 8 muscles from the lower extremity and trunk muscles . as a result of the analysis , all of the lateral gastrocnemius , soleus , tibialis anterior , vastus lateralis , biceps femoris , rectus abdominis , erector spinae , and trapezius upper fiber muscles showed the most statistically significant differences in the muscle activation effect , which was dependent on the intensity of the wbv stimulation . the volume , which is the amplitude factor , tended to have higher muscle activation at 80 than at 50 . , we could see that the muscle activation effect became greater as the volume increased and the frequency decreased . unlike the preceding studies , however , the muscle activity did not increase in direct proportion to the increase in vibration frequency . the muscle activity had the highest value at 10 hz , but it was reduced at 40 hz12 . both the excitatory inflow and the resultant muscle activity increase in the early stages as the vibration frequency increases ; however , the inhibitory inflow predominates and the muscle activity is lowered as the muscle tone increases . as a result , the greatest muscle activity emerges in the balanced frequency between the excitatory inflow and the inhibitory inflow , so the muscle activity is maximized at a specific frequency less than 25 hz ; in this range , muscle activity was reduced in this study13 . when considering each specific body part assessed in this study , the muscle activity of the lower extremities was generally higher than that of the whole body . we could see that the vibration stimulation from the soles of feet provided a wbv effect , in sequence , in the lower legs , thighs , and trunk ; however , this effect became weak as the stimulation proceeded to the upper part . this disadvantage might be compensated through changes in various postures ; however , the fact that the muscle activation effect was observed while subjects were standing is meaningful in itself , as it highlights the comfortableness and safety of these exercises the biggest advantages of wbv exercises14 , 15 . in the preceding studies , this seems to be due to the fact that some of the vibration energy is absorbed , passing through each joint and affecting the response of nerve root in the upper muscle . in conclusion , we can see that the wbv stimulation discriminatorily affects the entire body and , consequently , it provides a muscle activation effect . since muscle activity differs according to frequency , and given that this will result in variable exercise effects , a greater exercise effect will be obtained if diverse motions ( rather than the standing posture ) are combined . in addition , it is estimated that the different levels of intensity that were applied during detailed studies that aimed to assess the maximum activity for each individual muscle in each frequency band will be of importance to maximize the effects of wbv exercises .
[ purpose ] the purpose of this study was to investigate the effects of a whole - body vibration exercise , as well as to discuss the scientific basis to establish optimal intensity by analyzing differences between muscle activations in each body part , according to the stimulation intensity of the whole - body vibration . [ subjects and methods ] the study subjects included 10 healthy men in their 20s without orthopedic disease . representative muscles from the subjects primary body segments were selected while the subjects were in upright positions on exercise machines ; electromyography electrodes were attached to the selected muscles . following that , the muscle activities of each part were measured at different intensities . no vibration , 50/80 in volume , and 10/25/40 hz were mixed and applied when the subjects were on the whole - vibration exercise machines in upright positions . after that , electromyographic signals were collected and analyzed with the root mean square of muscular activation . [ results ] as a result of the analysis , it was found that the muscle activation effects had statistically meaningful differences according to changes in exercise intensity in all 8 muscles . when the no - vibration status was standardized and analyzed as 1 , the muscle effect became lower at higher frequencies , but became higher at larger volumes . [ conclusion ] in conclusion , it was shown that the whole - body vibration stimulation promoted muscle activation across the entire body part , and the exercise effects in each muscle varied depending on the exercise intensities .
You are an expert at summarizing long articles. Proceed to summarize the following text: protein phosphorylation is the most studied posttranslational modification that controls the dynamic behaviors and decision processes in cells of various organisms . in recent years , large - scale studies on protein phosphorylation based on mass spectrometry have been conducted on different organisms . some of them were carried out in plants ( 68 ) . as a result , a number of phosphorylation databases emerged , most of which focus on mammalian and prokaryotic systems . phospho.elm ( 9 ) contains verified eukaryotic phosphorylation sites , but most are from mammals . phosida ( 10 ) contains large - scale phosphorylation data in homo sapiens , bacillus subtilis and escherichia coli . plantsp ( 11 ) contains phosphorylation data on a few different plants , but it focuses on the annotation of plant protein kinases and protein phosphatases . phosphat ( 12 ) provides a database of phosphorylation sites collected from current literature solely for the model organism arabidopsis thaliana . pdb is unique in that it provides a resource of protein phosphorylation sites from various plant sources and contains multiple embedded search capacities for querying the database . by collecting and annotating plant phosphorylation data from different plant sources in a single database as a one - stop shop , we anticipate pdb that will serve as a useful resource not only for molecular biologists to study protein phosphorylation in plants and nonplant systems by comparison , but also for bioinformaticians to develop computational prediction tools on protein phosphorylation . reston ) developing seed obtained using a combination of data - dependent neutral loss and multistage activation on an ltq linear ion trap liquid chromatography tandem mass spectrometry system . details on the experimental design , which are available on the website ( pdb v1.0 release note ) , and the associated results and data analysis will be published elsewhere ( agrawal et al . , unpublished results ) . the dataset includes 14 670 nonredundant phosphorylation sites ( 8350 phosphoserine sites , 4750 phosphothreonine sites and 1567 phosphotyrosine sites ) from 6382 substrate proteins , representing the largest collection of plant phosphorylation data to date . experimental details about each phosphopeptide , such as charge state , cross - correlation score , peptide probability , spectrum count , spectrum plot , etc . , are available in the database . more protein phosphorylation data are being deposited into this database from recently completed large - scale studies of a. thaliana ( columbia ) and soybean ( glycine max var . submitted data will be displayed according to the current database format with full credit given to the submitting investigators . protein phosphorylation data are stored in a mysql relational database . with a php - based web graphical interface , the phosphorylation data in the database are downloadable , browsable and searchable . a user can browse the annotated phosphoproteins by organisms or by gene ontology categories ( 13 ) . a user can search for phosphoproteins by protein identifiers ( ncbi gi numbers , uniprot accession numbers or refseq accession numbers ) or protein descriptions , and search for phosphopeptides by peptide sequences . the main page of the search result lists all phosphoproteins / peptides meeting the searching criteria and gives some brief information , such as protein accession , protein description , source organism , consensus score , spectrum count , etc . the user can sort the result table according to different criteria , e.g. sort the phosphoproteins according to spectrum count from high to low . from the search result page the phosphoprotein page gives the details on the substrate protein , including the protein sequence with phosphorylation sites linked . clicking on a phosphorylation site will display its detailed information , such as its surrounding amino acids ( + /10 ) and a list of phosphopeptides that contain this phosphorylation site . clicking on show details presents the information about the peptide and clicking on more takes the user to the phosphopeptide page which contains additional information about the peptide . another useful feature on the website is the phosphopeptide blast utility as shown in figure 1 . by uploading a protein sequence as in figure 1a and querying it against the database using blast , a user can identify all the peptides in the query sequence that match one or more phosphopeptides in the database ( according to a user - defined e - value cutoff ) . in the blast result page as figure 1b , the blast alignments are displayed with links to the phosphopeptides and phosphorylation sites . in addition , to graphically representing phosphopeptide blast results , we developed a tool to view phosphopeptide blast results . figure 1c shows one example after submitting a phosphopeptide blast result to this tool by clicking on send to phosphopeptide blast browser in figure 1b . all the blast alignments are displayed with an e - value color scheme so that the user can know how similar the peptides in the query sequence are to the phosphopeptides . in addition , each residue in the query sequence that is aligned to one or more phosphorylation sites in the matching phosphopeptides is explicitly colored and hyperlinked , if it is serine , threonine or tyrosine . a user can also submit a protein query sequence directly to this tool under the the blast utility and blast result browser does not aim to explicitly predict phosphorylation sites or phosphorylation motifs in the query protein sequences , but does help the user to gain some related biological meaning about the query sequences . for example , if a user has a human phosphoprotein in hand and is interested to know whether similar phosphopeptides exist in plants , he / she may find this tool useful . alternatively , if a user wants to know whether a plant protein contains phosphorylation sites , this tool may help him / her to gain some knowledge of the empirical evidence for phosphorylation based on the related sequences from the database in a conservative or semi - conservative manner . figure 1.phosphopeptide blast example ( query sequence : arabidopsis atp binding protein ; dataset : oilseed rape phosphopeptides ) . ( a ) user interface for inputting a query protein sequence and selecting e - value threshold . the matching phosphopeptides are hyperlinked to the corresponding phosphopeptide pages which show their detailed information . the peptide sequences are rendered with different colors to show the different e - values of hits as indicated in the blast e - value color scheme legend . each phosphorylation site in each phosphopeptide is also hyperlinked and colored as blue , and so is the corresponding residue in the query sequence if it is y(tyrosine ) . phosphopeptide blast example ( query sequence : arabidopsis atp binding protein ; dataset : oilseed rape phosphopeptides ) . ( a ) user interface for inputting a query protein sequence and selecting e - value threshold . the matching phosphopeptides are hyperlinked to the corresponding phosphopeptide pages which show their detailed information . the peptide sequences are rendered with different colors to show the different e - values of hits as indicated in the blast e - value color scheme legend . each phosphorylation site in each phosphopeptide is also hyperlinked and colored as blue , and so is the corresponding residue in the query sequence if it is deposit phosphorylation datasets from large - scale studies of a. thaliana and soybean , which are in the process of being annotated.integrate more plant phosphorylation datasets from other investigators into pdb and continue updating the database with new advances in mining and prediction analysis of plant phosphorylation.integrate information on phosphorylation motifs and protein kinase specificity.integrate additional information on protein phosphorylation data , such as pfam domain , cross - species conservation data , pathway information , etc.improve the current utilities and implement more tools , such as advanced search tool for querying by a user - defined combination of different criteria.predict protein structures of phosphoproteins and highlight phosphorylation sites in a web - based protein structure viewer . deposit phosphorylation datasets from large - scale studies of a. thaliana and soybean , which are in the process of being annotated . integrate more plant phosphorylation datasets from other investigators into pdb and continue updating the database with new advances in mining and prediction analysis of plant phosphorylation . integrate information on phosphorylation motifs and protein kinase specificity . integrate additional information on protein phosphorylation data , such as pfam domain , cross - species conservation data , pathway information , etc . improve the current utilities and implement more tools , such as advanced search tool for querying by a user - defined combination of different criteria . predict protein structures of phosphoproteins and highlight phosphorylation sites in a web - based protein structure viewer . national science foundation ( grant number dbi-0604439 to j.t . ) . funding for open access charges : national science foundation .
p3db ( http://www.p3db.org/ ) provides a resource of protein phosphorylation data from multiple plants . the database was initially constructed with a dataset from oilseed rape , including 14 670 nonredundant phosphorylation sites from 6382 substrate proteins , representing the largest collection of plant phosphorylation data to date . additional protein phosphorylation data are being deposited into this database from large - scale studies of arabidopsis thaliana and soybean . phosphorylation data from current literature are also being integrated into the p3db . with a web - based user interface , the database is browsable , downloadable and searchable by protein accession number , description and sequence . a blast utility was integrated and a phosphopeptide blast browser was implemented to allow users to query the database for phosphopeptides similar to protein sequences of their interest . with the large - scale phosphorylation data and associated web - based tools , p3db will be a valuable resource for both plant and nonplant biologists in the field of protein phosphorylation .
You are an expert at summarizing long articles. Proceed to summarize the following text: human infection comes from eating undercooked meat , especially the snake or frog , or ingesting water contaminated with infected cyclops . clinical manifestations are diverse , including non - specific discomfort , vague pain , palpable mass , headach , or even no symptoms . most infection is presented with lumps in soft tissues , such as subcutaneous tissues or muscles in healthy adults [ 2 - 4 ] . in some cases , it is developed among immune - compromised patients , including aids patients , renal allograft recipients and patients with advanced solid tumors [ 5 - 8 ] . herein , we report a case of human sparganosis manifested overtly and mimicking myeloid sarcoma in a patient undergone allogeneic hematopoietic stem cell transplantation ( hsct ) for treatment of myelodysplastic syndrome ( mds ) . a 56-year old male visited our out - patient clinic for pancytopenia . bone marrow biopsy and karyotype analysis showed myelodysplastic feature with 14% blast count and normal karyotype , indicating mds , refractory anemia with excessive blast . he received 4 cycles of decitabine treatment during searching for matched unrelated donor , and partial response was shown . allogeneic peripheral hsct with myeloablative conditioning regimen consisting of busulphan and cyclophosphamide was performed from a hla full - matched unrelated donor . however , at day 5 post - hsct , 5 cm - sized hard and movable mass in the left scrotum and multiple palpable lymph nodes was detected at the left inguinal area . pelvic magnetic resonance imaging and computed tomography were done and it was suspected initially as a myeloid sarcoma in the subcutaneous fat layer of the left scrotum and pathologic lymphadenopathy at the left inguinal area ( fig . gun biopsy was done on the left scrotal mass to avoid bleeding due to thrombocytopenia after hsct . the result of the gun biopsy was inflamation of fibrovascular soft tissues with extensive interstitial eosinophilic infiltrations without evidence of malignancy . subsequently , igg antibody tests for paragonimiasis , cysticercosis , sparganosis , and clonorchiasis were done , and the results were positive for sparganosis and negative for all others . we re - evaluated the patient 's past history and found out that he had swam in the river and ate raw fish , cow liver , and pork meat frequently when he was young . for a next step , the cut surface of the biopsied specimen showed a cyst filled with whitish and myxoid tissues , measuring 2.00.6 cm ( fig . the biopsy result was severe acute and chronic inflammation and fibrosis with a sparganum worm ( fig . he was discharged after complete wound healing and stabilization of the post - hsct condition . most sparganosis cases present as lumps in subcutaneous tissues or intermuscular fascia , which are non - tender and sometimes resolve spontaneously . therefore , the diagnosis is often made incidentally by an imaging study or surgical biopsy in many cases . because of late diagnosis , the sparganum worm is presumed to have lived many years , and even a 10-year survived sparganum was reported . the final diagnosis is done by the surgical biopsy proving the presence of the worm . however , immunodiagnosis is also recommended to establish a preoperative diagnosis when soft tissue tumors are detected in patients living in endemic areas . sometimes , mass - like lesions of sparganosis are confused as solid tumors or other benign diseases , such as varicose vein , whereas there have been no reported cases mimicking myeloid sarcoma [ 8 - 11 ] . in our case , the left inguinal movable mass was initially suspected as a myeloid sarcoma , which is the extramedullary manifestation of acute leukemia , by initial imaging techniques . however , the positive anti - sparganum igg antibody test after a gun - biopsy suggested sparganosis rather than malignancy . there were few reports of sparganosis developed in immune - compromised patients , including malignancy , aids , and renal allograft recipients [ 5 - 7 ] . in our case , the immunosuppressive condition of transplantation might have brought out the hidden clinical presentation of the pre - existing sparganum infection . however , the causality between this condition and the development of sparganosis is unclear because the time of mass development from transplantation was too short , and the possibility of sustained sparganosis before transplantation also existed . this case suggests that physicians have to give an effort to exclude sparganosis with careful diagnostic approaches , including a detailed history taking , serologic test for parasites , optimal image techniques , and tissue obtainment when they face mass - forming lesions in patients under these clinical situations .
we report here a case of inguinal sparganosis , initially regarded as myeloid sarcoma , diagnosed in a patient undergone allogeneic hematopoietic transplantation ( hsct ) . a 56-year - old male patient having myelodysplastic syndrome was treated with allogeneic hsct after myeloablative conditioning regimen . at day 5 post - hsct , the patient complained of a painless palpable mass on the left scrotum and inguinal area . pelvic magnetic resonance imaging and computed tomography revealed suspected myeloid sarcoma . gun - biopsy was performed , and the result revealed eosinophilic infiltrations without malignancy . subsequent serologic igg antibody test was positive for sparganum . excisional biopsy as a therapeutic diagnosis was done , and the diagnosis of sparganosis was confirmed eventually . this is the first report of sparganosis after allogeneic hsct mimicking myeloid sarcoma , giving a lesson that the physicians have to consider the possibility of sparganosis in this clinical situation and perform adequate diagnostic and therapeutic approaches .
You are an expert at summarizing long articles. Proceed to summarize the following text: the obtained crystal structures of tyrosine - phosphorylated stat1 and stat3 demonstrated that interaction of two nds within one stat dimer is unlikely . these observations suggested that the nds are free to promote other protein - protein interactions . in particular , two stat dimers bound to adjacent gas elements may form a stat tetramer via nd - nd interaction . such cooperation in dna binding via nds allows fine - tuning of transcriptional responses through selective binding of different stat proteins on the promoters containing multiple stat binding sites and through binding to weak stat - binding sites . so far , the nd of stat1 , stat4 , stat5 and somewhat stat3 were found to form tetrameric complexes , at least on selected promoters . crystallographic studies identified invariant w37 as essential for the nd dimerization . other amino acid residues ( q36 , t40 and e66 ) were predicted to be involved in interactions between -helices within the nd . however , subsequent mutational analysis of the stat1 and stat4 nds demonstrated that these residues are unlikely to mediate interactions at proposed interface , and suggested an alternative dimer interface that involves s12 , l15 , dr19 ( -helix2 ) and f77 and l78 ( -helix 7 ) . deletion of the nd or the mutation in critical w37 residue responsible for nd dimerization resulted in abrogation of tetramer formation and transcriptional stimulation . the mutation of a single f77 residue in the nd of stat1 was recently found to preclude both the dephosphorylation and the oligomerization of stat1 dimers . vinkemeier and meyer have shown the influence of defective oligomerization on a complex phenotype such as the induction of an antiviral state . they found that the antiviral protection conferred by ifn was strongly reduced , whereas the ifn response was not measurably affected . nd - mediated stat5 tetramerization was found to be essential for il-2-induced regulation of the il-2 response element in the human il-2ra gene . the functional importance of tetramer formation was revealed by the decreased levels of transcriptional activation associated with hypomorphic mutations in n - terminal residues . in case of stat4 , substitution of w37 with alanine unexpectedly prevented ifn-induced tyrosine phosphorylation of stat4 monomer , blocking both dimer and tetramer formation . the requirement of the stat4 nd for stat4 activation was confirmed for il-12 signaling using stat4-deficient transgenic mice that express human full - length stat4 or n - terminal deletion mutant . while full - length stat4 rescues il-12 responsiveness , the stat4 n - terminally truncated protein does not undergo phosphorylation and therefore t - cells expressing this mutant do not undergo proliferation . the requirement for stat tetramerization via nd may contribute to selective activation of certain genes expression . for example , tetramerization of stat3 is required for the formation of enhancesomes on the promoter of 2-macroglobulin , but it is dispensable for il-6-induced activation of socs3 , which only requires stat3 dimer binding to the promoter . stat5 tetramerization is necessary for activation of il-2ra expression , but is dispensable for -casein . moreover , despite high homology between nds of various stats , each domain has specific functions that may , at least partially , define precise regulation of stat proteins functions . for example , substitution of stat4 nd with that of stat1 results in inability of chimera protein to undergo ifn-induced tyrosine phosphorylation and to bind dna probes in emsa assay . also , substitution of the stat1 nd with that of stat4 failed to restore ifn-induced mhc class i expression in u3a cells , despite the ability of this chimera to form emsa complexes similar to those of wild - type stat1 . this data suggests that the nds of stat1 and stat4 are not fully interchangeable for gene - specific transactivation events . it is thus conceivable to disrupt functions of various stats using selective inhibitors of nds . recently , stat5a - stat5b double - knock - in nd mutant mice in which stat5 may form only dimers but not tetramers were generated . in contrast to stat5-deficient mice that exhibited perinatal lethality , nd mutant mice were viable but had fewer cd4cd25 t cells , nk cells , and cd8 t cells , with impaired cytokine - induced and homeostatic proliferation of cd8 t cells . the observation suggested that stat5 dimers were sufficient for survival and for regulation of some target genes , and that tetramerization of stat5 was only critical for cytokine responses and normal immune function . the data obtained from double - knock - in mouse model agreed with previous report on the critical role of the stat5 nd in human stem cells maintenance and repopulating activity . therefore , the stat5 nd has an essential function during normal physiological development of immune system . it has been found that stat5 exists as a tetramer in cancer cells of 25% patients with leukemia , while this was not observed in normal human bone marrow or peripheral blood cells . in addition , stat5 tetramer formation resulted in stronger and larger dna binding complexes compared with those formed by the dimers . mutations in the stat5a nd abolished tetramer formation and prevented induction of leukemia due to inability of nd - mutated stat5a to rescue stat5 t cell proliferation , despite the persistent activation of stat5n proteins . these observations have proven that the enhanced tetramer formation through nd is the essential feature responsible for leukemogenesis . one of the mechanisms by which tetramer formation may contribute to leukemogenesis is increased occupancy of weak sites to a threshold required for transcriptional activity , which together with the greater degree of flexibility in dna sequence tetramer recognition was suggested to widen target gene spectra . stat5 target genes that control apoptosis , cell cycle progression , and proliferation , such as cyclin d3 , bcl - xl , bcl-2 , osm , cd25 , cis , socs-2 , als and igf-1 , contain at least two stat5 binding sites in their regulatory regions and their expression is controlled by stat5 tetramer . it has been established that stat5 lacking the nd ( stat5a-n ) can not protect c - kitlin - sca-1 cells from apoptosis or induce bcl-2 expression . signaling as an achilles heel of persistent stat5 activation and highlighted the potential therapeutic importance of targeting stat5 nd - mediated regulation of bcl-2 family members . although the significance of tetramerization vs. dimerization still remains to be established for other stat proteins , in particular for stat3 , the importance of the nd in cancer cells was confirmed by a study in which peptides inhibitors targeting the nds of stat3 or stat5 caused growth inhibition in breast cancer cells . therefore , interference with stats tetramerization through the nd may be an effective therapeutic strategy for cancer treatment . the finding that stat4 nd is essential for activation by cytokine receptors led to an assumption that nd dimerization of unphosphorylated stat4 is a pre - requisite for stat4 phosphorylation and transcriptional function . the yeast two - hybrid analysis of nd interactions , where the nd of each stat protein was expressed in the pfbl23 and gadt7 vectors to explore nds as baits for all other nds ( prey ) , demonstrated that all stat nds are involved in homotypic dimerization . interestingly , nds of stat5a and stat5b that differ only by 11 amino acid residues out of total 130 still showed only selective homotypic dimerization , and did not demonstrate any cross - reactivity . these data indicate that in addition to stabilizing tetramer formation , stat nds may have an important role in dimerization of non - phosphorylated stat proteins . however , the significance of this pre - association is not completely understood . in case of stat4 , such dimer formation may enhance presentation to receptor - jak complexes favoring synchronized phosphorylation of the two monomers and allowing formation of active stat dimer by simple intramolecular rearrangement . dimerization of unphosphorylated stat1 strongly depends on the nd because its deletion increased the dissociation constant 100-fold , from 50 nm to 34 m . crystallographic studies of stat1 demonstrated that the structure of each nonphosphorylated monomer is identical to phosphorylated stat1 monomer , however , the monomers in the non - phosphorylated protein are arranged differently , and the nd interactions are essential for an antiparallel stat1 dimer structure . a deletions of nd or mutations disrupting the stat1 nd dimerization ( m28 , f77 and l78 ) did not affect stat1 ability to undergo phosphorylation in response to ifn or ifn and form tyrosine - phosphorylated dimers , although such stat1 mutants did not possess the transcriptional activity . stat1 nd appears to regulate association with the nuclear phosphatase tc45 and subsequent stat1 dephosphorylation . indeed , deletion of the n - terminal domain of stat3 abrogated dimer formation , as shown by bnpage and 2f - fcs . however , the homotypic interaction of the n - terminal domain of stat3 are of low affinity ( 3.7 mm ) compared with that of stat1 ( 23 m ) and stat4 ( 2.7 m ) . point mutations analogous to those that disturb homotypic interaction of the n - terminal domain of stat1 had no detrimental effect on the dimerization of stat3 . therefore , the n - terminal domain of stat3 might not contribute to stat3 dimerization by homotypic interaction but by reciprocal interactions with another domain of stat3 . the sh2-domain could be a candidate for an interaction with the n - terminal domain because it has been shown that mutation of the sh2-domain affects dimer formation of unphosphorylated stat3 . such an interaction would lead to an antiparallel orientation of the latent stat3 dimer , in contrast to the parallel orientation of the activated stat3 dimer . however , it should be noted that concentration of unphosphorylated stat3 in jurkat cells stimulated with il-6 is about 100-times higher than stat1 ; therefore , it is possible that despite low affinity of the stat3 nd interactions they are biologically relevant . deletion of the stat3 nd does not impair il-6-dependent tyrosine phosphorylation , nuclear import or dephosphorylation kinetics , indicating that this region is not essential for stat3 recruitment to the il-6 receptor complex , translocation to the nuclear compartment or downregulation . however , the phosphorylated stat3 dimers lacking the n - terminal domain do not accumulate in the nucleus.a similar contribution of the n - terminal domain to nuclear accumulation has been shown for stat1 . these findings point to a functional role of the n - terminal domain in nuclear import of activated stat3 that deserves further investigation . the deletion of the stat5a nd also does not abrogate cytokine - induced tyrosine phosphorylation , dimerization or dimer dna binding . however , such deletion appears to render constitutive activation , indicating nd s negative regulatory function . interestingly , the nd truncated stat5a / b are predominant isoforms binding to dna in prostate cancer cells . the authors convincingly demonstrate that processing takes place in vivo , but not in vitro during the sample preparation . however , the exact mechanisms of proteolytic stat5a / b cleavage in prostate cancer cells has not been deciphered and the enzymes responsible for it have not been identified . considering that pias3 interacts with stat5 nd to repress stat5-dependent transcription , this modification may represent a molecular mechanism by which stat5a is able to evade inhibition of signaling by pias3 in human prostate cancer cells . in contrast , breast cancer cells , like mcf-7 and t47d , contain full - sized stat5a / b only . prolactin - stimulated activation is efficiently inhibited by pias3 , suggesting different mechanisms of regulation of stat5a / b activity in breast and prostate cancers . it is not known at present whether other stat proteins undergo the n - terminal proteolitic cleavage . identification of the proteases responsible for generation of the short form stat5a / b in prostate cancer may present new therapeutic targets . regulation of gene expression by unphosphorylated stats ( u - stat ) may constitute another potential role of the nd . the extensive investigation from stark s laboratory documented that both u - stat1 and u - stat3 play important roles in the regulation of gene expression . it has been proposed that u - stat1 binds to dna as a monomer by contacting one half of a palindromic gas motif , or forms dimers that also allows to bind gas sequences in vitro , though with much lower affinity , compared with the phosphorylated stat1 dimer . u - stat1 crystal structure suggests that unphosphorylated dimers bound to dna are likely to be formed via the nd interactions . most of the u - stat1 dimers exist in antiparallel conformation , but a small proportion of unphosphorylated stat1 adopts the parallel conformation of activated stat1 . we recently observed that u - stat3 also can bind to gas sequences both as a dimer and as a monomer , consistent with previous observations for u - stat1 . interestingly , atomic force microscopy allowed for detection of dimers of different shapes suggesting that u - stat3 dimers may bind dna in both parallel and anti - parallel conformation . the significance of the nd interactions for u - stat3 binding to dna and their role in regulation of gene expression remains to be investigated . if proven important for driving expression of genes regulated by u - stat3 , the disruption of nd - based dimerization may be a powerful tool to inhibit stat3 functions for therapeutic purposes . despite the importance of tetramer formation for gene expression , and potentially for tumorigenesis , it is probable that nd is also involved in controlling gene expression through interaction with other binding partners . the human protein reference database ( www.hprd.org ) lists 102 binary interactions for stat3 , 77 for stat1 , 13 for stat2 , 11 for stat4 , 53 for stat5a , 42 for stat5b and 18 for stat6 . the differences in the numbers of identified interactions reflect the level of popularity of a particular stat in research community rather than intricacy of its interactions . the diversity of interactions is a strong indicator of the complexity of their function s regulation . the list is far from being complete and includes only interactions for which interacting domains of stat3 have been identified . binary interactions of stats with other proteins are considered among the most biologically appealing yet chemically intractable targets for drug discovery . only for several proteins domains that are involved in interactions the nd of phosphorylated stat proteins is exposed on the surface of dimers or tetramers as suggested by crystallographic data and as recognized by native dna binding assays ( emsa ) or antibody supershift experiments . the location of the interacting stat3 domain is color - coded . in the cases where interacting domain is not localized precisely or interaction involves two domains , gradient coloring is used . nd , n - terminal doman ; cc , coiled - coil domain , dbd , dna - binding domain , ld , linker domain , sh2 , sh2-domain , tad , transactivation domain . during last few years , it has been understood that the nds of stat proteins undergo post - translational modifications that control the repertoire of stat protein - protein interactions . it has been shown that stat nds may be phosphorylated , acetylated , methylated , and glycosylated . arginine 31 residue is known to undergo methylation and is conserved across stat family members . although the work describing this finding has been criticized , a recent review on jak - stat pathway raises a voice in support of the role of r31 methylation in regulation of stat1 interactions with pias1 which catalyzes sumoylation of lysine residues and stat1 transcriptional activity . several recent reports have shed the light on protein - protein interactions that involve stat3 nd , including interactions with hdac1 and ape1 . two lys residues , 49 and 87 in the stat3 nd , are acetylated by p300 . lys - to - arg point mutations ( stat3 k49r / k87r ) that blocked p300-mediated stat3 acetylation had no effect on inducible dna binding , but abrogated il-6-induced angiotensinogen expression . although stat3 k49r / k87r rapidly translocated into the nucleus , it did not bind p300 and had delayed cytoplasmic redistribution . stat3 was also found to interact with hdac1 through the nd , which resulted in deacetylation of the domain and repression of stat3 transcriptional activity . these findings indicated that acetylation - deacetylation of stat3 provides another signaling axis to control the il-6-stat3 pathway in addition to phosphorylation - dephosphorylation . the follow - up study from the same group demonstrated that only acetylated stat3 forms an inducible complex with the apurinic / apyrimidinic endonuclease 1 ( ape1)/redox effector factor-1 ( ape1/ref-1 ) , an essential multifunctional protein in dna base excision repair in response to il-6 . ape1 selectively binds nd , and this interaction is required for stat3 stable chromatin association with the promoters of suppressor of cytokine signaling-3 ( socs3 ) and -fibrionogen . stat5 n - terminal domain interacts with the glucocorticoid receptor , which can control gene expression as either a coactivator or corepressor . the stat5 nd undergo glycosylation on t92 that is crucial for binding to the coactivator of transcription creb - binding protein and eventually p300 that are established coactivators of gene expression . in addition , phosphositeplus lists a number of post - translational modification of the stat nds that were only detected by mass spectrometry analysis . remarkably , no modifications were detected in the nds of stat2 and stat4 , while stat1 , stat3 and stat5a / b nds undergo phosphorylation , acetylation and ubiquitinylation . even without understanding the precise role of detected modifications , we may speculate that they regulate stat s protein - protein interactions that result in changes in stat functionality . it has been shown that stat3 can play opposing roles in cellular transformation depending on the genetic background of the tumor . one example includes induction of a highly aggressive t cell leukemia in mice by activated stat3 but prevention of c - myc - induced transformation of mouse embryonic fibroblasts deficient for p53 . another example is tumor - suppressive function of stat3 in glioblastomas deficient in tumor suppressor pten , and oncogenic functions in glioblastomas that express nuclear epidermal growth factor receptor type iii variant ( egfrviii ) . there is a substantial heterogeneity in genetic backgrounds of tested cell lines , therefore it is not surprising that there is also a heterogeneity in responses to inhibition of stat3 signaling . it is tempting to speculate that nd protein - protein interactions are responsible , at least partially , for switching of stat3 function from pro - apoptotic to pro - survival during cell transformation . we found that inhibition of the stat3 nd had little effect on normal epithelial cells , while it induced fulminate apoptosis in breast and prostate cancer cells . these data suggested that the stat3 nd performs different functions in cancer as compared with that observed under normal physiological conditions . one can speculate that different roles may be defined by involvement of the stat3 nd in various protein - protein interactions and possibly by differential post - translational modifications of the nd . the identification of differences in signaling events that underlie differential activity of the stat3 nd in normal and cancer cells may offer a potentially novel therapeutic target for cancer treatment . development of selective chemical probes and potential therapeutic agents for stat domains is challenging because the critical interacting surfaces appear to lack deep hydrophobic involutions that enable potent and selective targeting by small molecules . in addition , stats localization within the cell positions them beyond the reach of protein therapeutics . a considerable interest has therefore arisen in next - generation targeting molecules that combine the broad target recognition capabilities of protein therapeutics with the robust cell - penetrating ability of small molecules . we have been using successfully retro - inverso lipopeptides , one of novel classes of synthetic miniproteins with greatly improved pharmacologic performance , increased target affinity , proteolytic resistance and serum half - life while conferring on them high levels of cell penetration . it should be noted here that chemical biology offers very powerful tools in studying the function of certain parts of proteins that provide for much more solid conclusions than genetic methods , but only if the chemical probes are highly selective . experiments involving expression of stats mutants lacking certain domains or containing point mutation in stat - null cells are unlikely to generate the phenotype reflecting correctly the function of the mutant protein in stat - dependent cells . functions of stats are known to be cell - dependent and stat - null cells are unlikely to have the correct combination of partner proteins . we based the original design of stat nd inhibitors on the structure of stat4 n - terminal domain . dimerization of stat4 nd was well established and our original intention was development of inhibitors of dimerization . crystallography data suggested involvement of helixes 2 and 7 , while nmr data detected helixes 2 and 8 in the dimer interface . we originally made peptides corresponding to both helixes 2 and 8 and tested them for ability to interact with stat4 nd by nmr . peptide corresponding to helix 2 produced well defined changes in chemical shifts of stat4 nd , while peptide corresponding to helix 8 caused protein to precipitate , most likely due to unfolding . published characterization of dimerization propensity for different stat nds has shown that they differ significantly and that stat3 nd dimer is significantly less stable than stat1 and stat4 dimers thus highlighting the mechanistic differences in the way different members of stat family function . however , it should be noted that the levels of expression of different stats also differ by 100-fold , as was demonstrated for leukemia cells . higher concentration of stat3 may compensate for low affinity and result in less pronounced structural differences between stats . the study by wenta et al . also suggested the existence of two modes of nd dimerization , at least for stat1 . although the structural features of these two modes are unknown , it is possible that both models obtained from crystal structure and nmr studies of stat4 are correct and have indeed identified two naturally occurring interaction interfaces . nmr studies of nd inhibitors interaction with stat4 nd domain suggest that helix 2 analogs are likely to inhibit significantly more than just nd dimerization . changes in chemical shifts detected in the hsqc nmr spectrum of stat4 suggest that domain undergoes significant conformational changes upon binding of the peptide ( fig . 3 ) . it is interesting that the residues that are involved are localized mostly on one face of the domain , while the other half of it appears to be subjected to much lesser change ( fig . however , the changes cover significant fraction of the domain structure and thus many binary interactions of the domain can be affected . figure 3 . residues of stat4 nd ( in red ) with the most significant chemical shift perturbations detected upon binding of peptide corresponding to helix 2 ( ac - eikfleqvdkfy - penetratin ) . the data generated for stat4 has been used for the design and development of nd inhibitors of stat1 , stat3 and stat5 . structural studies suggest that n - terminal domains of stat proteins have very similar folds . tertiary structures of stat1 and stat4 nds to select initial lead analog of stat3 helix 2 for optimization . during optimization of peptide length and structure , however , our later findings suggested that simple fusion of the peptides to fatty acids was as effective as attachment of cell - penetrating peptides for intracellular delivery of compounds . extensive studies of structural and biological properties of lipopeptide mimetics of the conserved region of several important but non - druggable molecular targets have revealed that membrane anchoring through the attachment of fatty acid chains can produce highly selective and potent inhibitors of the corresponding protein . membrane anchoring through lipidation contributes to high potency of compounds in three ways : ( 1 ) lipidation facilitates cell entry ; ( 2 ) fatty acid chain causes membrane insertion and concentrates the inhibitor near intracellular and plasma membrane , where practically all signaling events occur ; ( 3 ) membrane anchoring enables folding of otherwise unfolded protein fragment , which results in an increase in potency , frequently by 2 to 3 orders of magnitude . although stabilization is due to enhanced interaction of amino acid side chains with the lipid bilayer , it does nt interfere with peptide s ability to interact . thus , it increases the time the peptide spends in the active conformation , rather than freezes it leaving sufficient time for the folded peptide to stick out of the membrane . currently , four lipopeptides ( daptomycin , liraglude , tesamorelin and caspofungin ) are used in the clinic . however , many are being developed , and their pharmacological properties make them very convenient chemical biology tools . the remarkable advantage of the approach is that it can be used for rational design of the probes even in the absence of the structural data for the target protein . selection of the stretches of amino acid sequences for mimicking can be based on the conservation during the evolution . the assumption here is that the highly conserved regions are involved in functionally essential protein - protein interactions and thus compounds mimicking them can function as dominant negative inhibitors of the corresponding interactions . we have used the approach successfully for the development of lipopeptide inhibitors of receptors signaling upstream from stats and other non - druggable targets . the conservation - based selection however was nt applicable to stat proteins because entire primary structures are conserved . . however , this is a very unusual case that also has an important message in it : entire structure of stat3 is likely to be important for protein function and there are numerous opportunities in affecting stat function through the development of probes mimicking different parts of stat proteins . although not every member of the family was characterized structurally , available data allows speculating that overall fold is well preserved in entire family and thus stat1 , stat3 and stat4 structural data can be used for identification of fragments suitable for development of potential dominant negative inhibitors of all stats . once the stretches for mimicking are identified , the design of the probes is straightforward . the major challenge is determination of optimal positions for fatty acids attachment and the optimal length of the mimicking sequence . here are some ground rules : the preferred lipid position is at the ends of secondary structure elements ; attachment of fatty acid to the side chains , such as -amino group of lys is more likely to result in active compounds . however , we did come across several exceptions , when derivatives with fatty acids attached to -amino group of the n - terminal amino acid were more active . for in vivo use , compounds can be converted into retro - inverso analogs composed on all - d amino acids . they tend to have a more rigid structure that is beneficial in majority but not all cases . however , shorter fatty acids are frequently sufficient and provide for better solubility of compounds . application of the approach to nds of stats allowed us to uncover previously underappreciated role of stat3 nd in tumor growth and stat1 nd in kidney development . the data shows that nds of stats are promising therapeutic targets and lipopeptide inhibitors have a potential to serve as effective therapeutic agents . the wealth of currently available data implies that targeting domains other than sh2 can be an effective way of modulating activity of stats for generation of chemical biology tools and potential therapeutic applications . the data generated for stat n - domains suggest that rationally designed lipopeptide mimetics of fragments of proteins involved in jak - stat signaling can serve as powerful tools in studying the molecular and cellular mechanisms of signaling . it is evident that the function of stat transcription factors is regulated by complex and thus far poorly understood mechanisms involving numerous protein - protein interactions . the latest data also indicate that members of stat family are likely to have multiple functions that they perform not only in the nucleus , but other cellular compartments . targeting protein - protein interactions with rationally designed probes will lead to significant advances in our understanding of molecular mechanisms of jak - stat signaling , pathological processes involving the pathway and consequently therapeutic approaches to controlling and redirecting the function of this important family of proteins .
most attempts to develop inhibitors of stat transcription factors target either activating phosphorylation of tyrosine residue or sh2 domains . however , all six domains of stats are highly conserved between the species and play important roles in the function of this family of transcription factors . stats are involved in numerous protein - protein interactions that are likely to regulate and fine tune transcriptional activity . targeting these interactions can provide plentiful opportunities for the discovery of novel drug candidates and powerful chemical biology tools . using n - terminal domains as an example we describe alternative rational approaches to the development of modulators of jak - stat signaling .
You are an expert at summarizing long articles. Proceed to summarize the following text: the basic description of laryngeal innervation involves the superior laryngeal nerve ( sln ) providing ipsilateral sensory innervation to the supraglottis and motor innervation to the cricothyroid muscle , while the recurrent laryngeal nerve ( rln ) provides sensory and motor innervation to all other laryngeal structures . however , anatomic studies suggest that there is significant overlap of motor and sensory innervation among branches of these nerves . cadaveric studies of human larynges suggest a high prevalence of anastomoses between the internal and external branches of the sln ; between the internal branch of the sln and the rln , known in some instances as galen 's anastomosis ; and finally , between the external branch of the sln and the rln , which has been termed the human communicating nerve ( hcn ) [ 13 ] . while cadaveric dissection studies have illustrated the complexity and prevalence of these nerve connections , their impact on laryngeal function is not well understood . visualization of these nerve anastomoses in intact , non - dissected larynges is challenging . to our knowledge , we report a case where an extralaryngeal variant of the hcn was identified and stimulated during routine thyroidectomy . the patient was a 56-year - old woman with an incidentally noted , 1.1 cm right thyroid nodule . this system employs surface electrodes on the endotracheal tube that detect and record electromyographic activity from the thyroarytenoid muscles . in addition , stimulation - evoked muscle action potential was recorded including latency , amplitude and waveform morphology . during dissection of the right superior pole , a nerve branch was noted originating from the ebsln anterior to the cricothyroid muscle and coursing across the superior pole of the thyroid gland . this nerve then joined the main branch of the rln prior to its laryngeal entry at the cricothyroid joint ( fig . stimulation of the ebsln , but not the hcn , produced a strong twitch of the cricothyroid muscle . stimulation of the hcn ( set 100 ) at a current intensity of 0.5 ma produced a triggered compound muscle action potential with a peak latency and a peak - to - peak amplitude of 5.0 ms and 887.3 v , respectively ( fig . stimulation of the rln at a current intensity of 0.5 ma produced a triggered compound muscle action potential with a peak latency and a peak - to - peak amplitude of 4.8 ms and 989.5 v , respectively ( fig . stimulation of the ebsln at a current intensity of 0.4 ma produced a triggered compound muscle action potential with a peak latency and a peak - to - peak amplitude of 5.4 ms and 35.6 v , respectively ( fig . the sln terminated in its usual internal branch ( ibsln ; sensory ) and external branch ( ebsln ) supplying motor fibers to the cricothyroid muscle . an extralaryngeal variant of the hcn was noted , connecting the ebsln to the rln prior to its laryngeal entry at the cricothyroid joint . figure 2:stimulation of the hcn with the dragonfly neuromonitoring system at 0.5 ma demonstrated a distinct waveform ( a ) , with an onset latency of 3.2 ms and a peak - to - peak amplitude of 882.1 v . this waveform was distinct from those produced by stimulation of the proximal rln ( b ) or ebsln ( c ) . the sln terminated in its usual internal branch ( ibsln ; sensory ) and external branch ( ebsln ) supplying motor fibers to the cricothyroid muscle . an extralaryngeal variant of the hcn was noted , connecting the ebsln to the rln prior to its laryngeal entry at the cricothyroid joint . stimulation of the hcn with the dragonfly neuromonitoring system at 0.5 ma demonstrated a distinct waveform ( a ) , with an onset latency of 3.2 ms and a peak - to - peak amplitude of 882.1 v . this waveform was distinct from those produced by stimulation of the proximal rln ( b ) or ebsln ( c ) . the actual existence of the hcn was initially quite controversial , based on several cadaveric microdissection studies performed in the 18th , 19th and early 20th centuries showing mixed results . a technique known as sihler 's stain , which clears soft tissue and counterstains nerves , has been useful for mapping fine nervous anastomoses in the human larynx that are difficult to discern with microdissection techniques [ 1 , 2 ] . in a cadaveric study using sihler 's stain , sanders et al . identified connections between the ebsln and the rln in 4 of 10 human larynges ; the connection was intralaryngeal in all cases , with a branch of the ebsln passing through the posterior surface of the cricothyroid muscle , then across the pyriform sinus before joining rln branches in the thyroarytenoid muscle . in a second study of 27 hemilarynges , the authors found ebsln / rln connections in 12 ( 44% ) . in one of these examples , the connection joined the ebsln to the anterior branch of the rln prior to its entry into the larynx and thyroarytenoid muscle , similar to the situation noted in our case . the finding of one extralaryngeal hcn among 27 hemilarynges suggests that the prevalence of this variant may be 34% . in a microdissection study of 90 human larynges , connections between the ebsln and the rln were noted in 68% , either at the point where the rln innervated the lateral cricoarytenoid muscle , or in the paraglottic space prior to the rln entry into the thyroarytenoid muscle . these studies suggest that the presence of the hcn may be relatively common , though most of these anastomoses occur within the larynx . outside of cadaveric studies , intraoperative identification of an extralaryngeal hcn has not been described . this is likely related to the tendency of most surgeons to avoid , rather than identify , the ebsln during thyroidectomy and other central neck surgeries . however , recent literature suggests that routine identification of the ebsln using loupe magnification and intraoperative neuromonitoring may decrease occult ebsln injury and improve postoperative voice outcomes [ 46 ] . it has been suggested that the hcn may be the vestigial nerve of the fifth branchial arch , which has not been identified by anatomists [ 1 , 2 ] . some authors have also speculated that the presence of the hcn and other nervous anastomoses within the larynx might explain the variability in position of a paralyzed vocal fold following complete injury or transection of the ipsilateral rln [ 1 , 2 ] . in our case , electrostimulation of the hcn produced enough motor activity to elicit a distinct waveform on the neuromonitoring system , suggesting that it contributes to thyroarytenoid muscle function . the hcn is a connection between the sln and the rln that may be relatively common . functionally , it may contribute to thyroarytenoid motor activity and may in part explain the variability in vocal fold function following injury of the rln . intraoperative identification of this variant is rare , since most of the anastomoses occur within the larynx . newer practice guidelines advocating for the routine identification of the sln during thyroidectomy may lead to intraoperative identification of more examples of this extralaryngeal variant of the hcn . this work was supported in part by funds from the department of veterans affairs , blsr&d and the pnc foundation ( u.d . ) .
the human communicating nerve ( hcn ) is a connection between the superior and recurrent laryngeal nerves that has been described in cadaveric studies . we report a case of an extralaryngeal variant of the hcn that was identified and stimulated intraoperatively during thyroidectomy . this appears to be the first case of intraoperative identification of this anatomic variant , of which the functional significance remains unclear .
You are an expert at summarizing long articles. Proceed to summarize the following text: the estimated prevalence of t. nativa in walruses ( 24% ) is lower than in polar bears ( ursus marinus ) ( 60% ) , but is much higher than what has been observed in other marine mammals ( 3,5,6 ) . as it is often eaten raw , walrus meat is the most frequent source of trichinellosis in the arctic ( 5,711 ) . after ingestion of infected meat , larvae rapidly develop into adult worms in the host intestine ( 12,13 ) . first - stage larvae ( l1 ) are shed and then migrate via the intestinal lymphatic vessels and blood circulation to the striated muscular cells ( 12,14 ) . since larvae of t. nativa are destroyed by heat ( in 1 minute at 67c ) , the consumption of properly cooked meat does not represent a risk of infection . on the other hand , in contrast to t. spiralis , t. nativa larvae resist long - term freezing . consequently , this method of preservation does not prevent transmission of t. nativa in people . infectivity of larvae decreases after meat fermentation , but their inability to survive in igunaq is still equivocal ( 15,16 ) . clinical diseases were found in people who had eaten meat containing 14 larvae per gram ( 5,7 ) . trichinellosis is classically characterized by an incubation phase of 620 days , followed by a short gastro - intestinal phase associated with the adult parasites . fever , myalgia , facial oedema , rash , increased muscle - enzyme level in blood and eosinophilia are common clinical findings associated with the migrating larvae ( 10 ) . a chronic gastro - intestinal syndrome , characterized by prolonged diarrhoea without prominent muscular signs , has also been described in inuit people ( 9,10 ) . clinical and serologic evidences suggest that the classical myopathic form occurs at first infection by t. nativa , while the chronic gastro - intestinal form affects previously sensitized individuals . both types are associated with occasionally severe , prolonged and debilitating forms of infection ( 10 ) . diagnosis of trichinellosis is confirmed by parasitological examination of muscular biopsies or by the detection of specific antibodies ( 17 ) . medical management relies on the administration of anthelminthics ( mebendazole or albendazole ) and glucocorticosteroids ( 17 ) . from 1982 to 1997 , 9 outbreaks of human trichinellosis linked to walrus consumption were documented in inuit communities of nunavik ( 3 ) . in the largest one in 1987 , clinical evidence of infection was found in 62% ( 42/68 ) of the persons tested among 88 consumers of an infected walrus who participated in the epidemiological investigation that was carried out at the time . it is believed that these events contributed to the decrease in walrus harvests during the 1980s in the affected communities . since walrus hunting was viewed as an economically , culturally and socially important activity , stakeholders agreed to collaborate to prevent human trichinellosis and to promote the walrus hunt . in response to the initial outbreaks ( 19821983 ) , a first approach to the problem was to educate community members on the importance of cooking walrus meat to prevent trichinellosis . this recommendation was not successful since it did not take into account the cultural specificity of the communities , including the taste of the community members for raw walrus meat ( 4 ) . first attempts to analyze walrus meat for the presence of t. nativa were conducted by sending samples to a southern laboratory , but delays between submission and the results were too long . from 1983 to 1987 , a limited number of samples were locally analyzed by trichinoscopy in a few local health clinics . this method was time - consuming , had a relatively low sensitivity , suffered from the high personnel turnover at the clinics and failed to prevent new outbreaks ( 4 ) . in 1991 , several partners , including the salluit municipal corporation , the makivik renewable resource development department , the inuulitsivik health centre and the laval university public health department decided to conduct and evaluate a local pilot project of trichinellosis prevention in salluit , one of the most affected communities ( 4 ) . the nunavik trichinellosis prevention program ( ntpp ) began in 1992 in salluit and was then progressively refined and offered to every walrus - harvesting community in 1996 ( 3 ) . as will be described below , the ntpp consists of a regionally based screening program for trichinellosis in harvested walrus to achieve disease prevention . the objective of the present communication is to describe and review the ntpp with a special focus on the interweaving of science and action involved in its development . the effects of this program on the incidence of trichinellosis in nunavik communities and its assets and limitations will also be discussed . the ntpp , which started in 1992 in its present form , is a community - based program , involving many local partners such as the nunavik public health department , the nunavik research centre ( nrc ) of makivik corporation , the kativik regional government ( through the hunter support program ) , the municipal authority of each participating community and the boat captains . they receive protocols and sampling kits ( including identification tags , protocols and data sheets ) through the nrc . they are asked ( a ) to securely tag all pieces of meat from each walrus to ensure future traceability , ( b ) to sample the entire tongue from each walrus harvested ( from 1992 to 1999 , a section of the base of the tongue , the cheek , the diaphragm and the intercostal muscles were collected ) , ( c ) to ship samples to the nrc by the next available flight to kuujjuaq , ( d ) to wait for the results before distributing the meat ( unless it is consumed well cooked ) and ( e ) to follow the instructions of health and municipal authorities when results are communicated ( 6 ) . the nunavik research centre , in charge of the analyses , delivers results within 24 hours after sample reception . the procedure follows a protocol developed in association with the canadian food inspection agency ( cfia ) from a validated method for detection of t. spiralis in pork and horse meat . the main differences with the latter method are the type of muscle analyzed , a decreased quantity of muscles tested and an increased digestion time ( 18 ) . it consists of an enzymatic digestion coupled with a double - funnel separatory procedure ( 18,19 ) . briefly , 10 g of muscle tissue from 5 walruses are pooled together and blended in 50100 ml of 1% hcl . thirty grams of granular pepsin ( 1:10,000 us national formulary ) are dissolved in the paste . in a beaker , hcl solution is added to the mix , until it reaches 3 l. the recipient is put on a magnetic stirrer and heated at 45c in an incubator for 90 minutes . after incubation , the solution is filtered through a 180 m - pore sieve and it is then allowed to settle for 30 minutes in a 4 l separatory funnel . the lower 150 ml are drained into a second 500 ml separatory funnel , diluted with 350 ml of warm tap water and left to settle for 10 minutes . the lower 25 ml are poured into a petri dish and examined under a stereomicroscope at 20 magnification ( 18 ) . if larvae are present , each walrus tongue is individually analyzed with the same protocol to identify the infected animal(s ) . a pcr assay is later conducted on larvae isolates at the cfia laboratory to verify which species of trichinella spp . are present ( 20 ) . from 1992 to 1999 , if 1 or more of the 4 targeted muscles were missing from an animal , the sample was declared unfit and of undetermined trichinella status . ( 2002 ) , a negative result from the tongue was sufficient to grant an overall negative trichinella status to an animal ( 18 ) . the results of the analyses are communicated to the nunavik public health officer who immediately contacts the municipal authorities to inform them and provide proper food - management advice . the recommendations are as follows : meat of uninfected animals may be eaten raw or undercooked ; meat of untested walruses should be thoroughly cooked before consumption ( no pink centre ) ; infected meat should not be given to dogs but should be destroyed ; igunaq prepared from uninfected animals can be eaten without risk of trichinellosis . each piece of tested and negative walrus meat should be marked to that effect . recommendations and educative content have been communicated to community members through various strategies , including video documentaries on television , messages on radio , pamphlets , articles in makivik 's magazine and direct communication in annual hunter meetings . passive surveillance of occurrence of human cases of trichinellosis in communities is the responsibility of local health services with the assistance of the nunavik public health department . information regarding confirmed and probable cases of trichinellosis has been gathered since 1980 through the quebec system for mandatory declaration of disease . a confirmed case is defined as follows : compatible clinical manifestations with one of the following conditions : ( a ) identification of trichinella in muscular tissue from biopsy or , ( b ) detection of specific antibodies by eia in 2 following sera collections 3 to 4 weeks apart . a probable case is defined as : compatible clinical manifestations in a person who has eaten the incriminated meat in an outbreak context where at least one case has been confirmed . the surveillance system is enhanced through ongoing medical education and transfer of laboratory results showing high levels of oesinophils ( > 10% ) in white blood counts to the public health physician , who then contacts the treating physician to ensure that trichinellosis is considered in the differential diagnosis . walrus hunters participate in the ntpp on a voluntary basis . they receive protocols and sampling kits ( including identification tags , protocols and data sheets ) through the nrc . they are asked ( a ) to securely tag all pieces of meat from each walrus to ensure future traceability , ( b ) to sample the entire tongue from each walrus harvested ( from 1992 to 1999 , a section of the base of the tongue , the cheek , the diaphragm and the intercostal muscles were collected ) , ( c ) to ship samples to the nrc by the next available flight to kuujjuaq , ( d ) to wait for the results before distributing the meat ( unless it is consumed well cooked ) and ( e ) to follow the instructions of health and municipal authorities when results are communicated ( 6 ) . the nunavik research centre , in charge of the analyses , delivers results within 24 hours after sample reception . the procedure follows a protocol developed in association with the canadian food inspection agency ( cfia ) from a validated method for detection of t. spiralis in pork and horse meat . the main differences with the latter method are the type of muscle analyzed , a decreased quantity of muscles tested and an increased digestion time ( 18 ) . it consists of an enzymatic digestion coupled with a double - funnel separatory procedure ( 18,19 ) . briefly , 10 g of muscle tissue from 5 walruses are pooled together and blended in 50100 ml of 1% hcl . thirty grams of granular pepsin ( 1:10,000 us national formulary ) are dissolved in the paste . in a beaker , hcl solution is added to the mix , until it reaches 3 l. the recipient is put on a magnetic stirrer and heated at 45c in an incubator for 90 minutes . after incubation , the solution is filtered through a 180 m - pore sieve and it is then allowed to settle for 30 minutes in a 4 l separatory funnel . the lower 150 ml are drained into a second 500 ml separatory funnel , diluted with 350 ml of warm tap water and left to settle for 10 minutes . the lower 25 ml are poured into a petri dish and examined under a stereomicroscope at 20 magnification ( 18 ) . if larvae are present , each walrus tongue is individually analyzed with the same protocol to identify the infected animal(s ) . a pcr assay is later conducted on larvae isolates at the cfia laboratory to verify which species of trichinella spp . are present ( 20 ) . from 1992 to 1999 , if 1 or more of the 4 targeted muscles were missing from an animal , the sample was declared unfit and of undetermined trichinella status . ( 2002 ) , a negative result from the tongue was sufficient to grant an overall negative trichinella status to an animal ( 18 ) . the results of the analyses are communicated to the nunavik public health officer who immediately contacts the municipal authorities to inform them and provide proper food - management advice . the recommendations are as follows : meat of uninfected animals may be eaten raw or undercooked ; meat of untested walruses should be thoroughly cooked before consumption ( no pink centre ) ; infected meat should not be given to dogs but should be destroyed ; igunaq prepared from uninfected animals can be eaten without risk of trichinellosis . recommendations and educative content have been communicated to community members through various strategies , including video documentaries on television , messages on radio , pamphlets , articles in makivik 's magazine and direct communication in annual hunter meetings . passive surveillance of occurrence of human cases of trichinellosis in communities is the responsibility of local health services with the assistance of the nunavik public health department . information regarding confirmed and probable cases of trichinellosis has been gathered since 1980 through the quebec system for mandatory declaration of disease . a confirmed case is defined as follows : compatible clinical manifestations with one of the following conditions : ( a ) identification of trichinella in muscular tissue from biopsy or , ( b ) detection of specific antibodies by eia in 2 following sera collections 3 to 4 weeks apart . compatible clinical manifestations in a person who has eaten the incriminated meat in an outbreak context where at least one case has been confirmed . the surveillance system is enhanced through ongoing medical education and transfer of laboratory results showing high levels of oesinophils ( > 10% ) in white blood counts to the public health physician , who then contacts the treating physician to ensure that trichinellosis is considered in the differential diagnosis . for the first 4 years the ntpp was implemented in only 1 community ( salluit ) , and then extended in 1996 to every community harvesting walruses . since the extension of the program , members from 5 to 9 communities out of the 14 communities in nunavik participated in the walrus hunt and the ntpp each year ( fig . 1 ) . number of communities involved in the nunavik trichinellosis prevention program from 1992 to 2009 . the number of communities hunting walrus varies according to year because of several factors , including weather conditions and availability of boats . an increase in the percentage of hunted animals sampled and tested was documented during the 12 first years after the implementation of the ntpp in 1992 , with almost all the harvested walruses being tested from 2000 to 2004 . since the total number of harvested walruses was not recorded from 2004 to 2008 , the percentage of tested animals during these years is undetermined , but is believed to have remained very high . the prevalence of trichinella in walrus varied from 0 to 12% with peaks in 1997 and 2001 . all positive walruses during those peaks were all harvested in eastern hudson bay . since 2001 , the community going at that location is going hunting every few years at a farther location in hudson strait . figure 2 presents the results of the screening for trichinella sp . larvae in walrus and walruses harvested each year . from 1992 to 2009 , 20 positive animals were found in the 694 walruses tested ( 2.9% ) . number of walruses harvested since 1974 and results of the screening for trichinella sp . in walrus since the beginning of the nunavik trichinellosis prevention program . the total number of walrus harvested is not available from 2004 to 2008 , but it is believed to be close to the number of walruses tested . the black line with open circles stands for the proportion of positive walruses . from 1982 to 2009 , a total of 15 trichinellosis incidents were recorded in 7 communities in nunavik , nine of which were linked to the consumption of walrus meat . five of these 9 walrus - associated incidents occurred prior to the implementation of the ntpp , over a period of 10 years . two incidents occurred at the beginning of the generalization of the ntpp and were linked to walruses that had not been tested under this screening program . one outbreak occurred in 1997 after the consumption of an infected walrus while the meat was still in quarantine pending the results of analyses . the 2 cases recorded in 2006 were infected during travel in the adjacent territory of nunavut after eating meat from a walrus not tested and associated with a large outbreak in that jurisdiction . other animals involved in cases of trichinellosis in nunavik are fox ( 1996 ) , polar bear ( 1998 ) and black bear ( 2004 ) . the epidemiological investigations were not successful in identifying trichinellosis sources in 3 cases ( 1999 , 2004 , 2009 ) the 2 main goals of the ntpp are to prevent human trichinellosis and to promote the walrus hunt . as shown in table i , no locally acquired walrus - linked trichinellosis outbreaks have occurred in nunavik since 1997 , even though infected walruses have been detected almost yearly since then ( fig . even if human exposures occurred in that event , the results of the analyses performed under the ntpp were communicated to the regional health services and were instrumental in the treatment of these consumers before the onset of clinical signs . ( 2002 ) , is the first report of a successful trichinellosis secondary - prevention initiative in the arctic ( 3 ) . only 2 other incidents of walrus - linked trichinellosis were recorded since the implementation of the ntpp , both during the period of generalization of the program to all walrus - hunting communities . the rare occurrence of walrus - linked trichinellosis since 1992 contrasts with the situation observed in the 1980s where outbreaks occurred every other year or so on average . in addition , it was noticed that walruses from some locations seemed to be more frequently infected . this observation is supported by the apparent decrease in prevalence associated with the avoidance of those locations by hunters . chronology and characteristics of individual trichinellosis incidents in nunavik from 1982 to 2009 epid : based on epidemiologic investigation ; lab : based on laboratory testing . the walrus involved in this outbreak was harvested and consumed in nunavut , outside the nunavik territory , and was not tested at the nrc prior to consumption . these inter - year changes are probably driven by variations in the availability of boats and financial resources , as well as weather conditions . due to this high annual variability , it is difficult to assess the impact of the ntpp on the walrus - hunting effort , even though the number of walruses harvested in nunavik appears to have stabilized over the last 10 years after an apparent decrease . nevertheless , in general , field workers and hunters feel that the ntpp has favoured the perpetuation of the hunt . the total cost of the program is difficult to evaluate since most of it is supported by in - kind participation of the different partners . nevertheless , because of the very high human and financial costs associated with the management of a single outbreak of trichinellosis in this remote and isolated arctic territory , the investment in the ntpp is considered economically sound . the ntpp can thus be considered as a successful and rational program , based on the absence of recent cases of trichinellosis acquired from walrus meat and the positive effect of the program on the continuation of the hunt . the motivation and empowerment of participating communities constitute another positive spin - off of the ntpp . first , the ntpp participates in the public mission of ensuring health in diverse ways : directly by trichinellosis prevention and indirectly because increasing food diversity is considered as a component of nutritional - diseases prevention ( 2,21 ) . the program also has positive impacts on the economic , social and cultural role of the hunt , especially because it values hunters as skilful food providers . this convergence of interest , shared by the inuit community members , the local and regional organizations and the public health authorities , is probably a major asset as regards the perpetuation of the ntpp ( 22 ) . in addition , it is recognized that effective community participation , which involves the sharing of responsibilities , contributes to the achievement of sustainable health objectives and could lead to the appropriation of research outcomes by the community ( 23,24 ) . the fact that the technical operations of the program are supported by an inuit entity ( makivik corporation ) with trained personnel ( wildlife specialists and laboratory technician ) actually facilitated communications and may have contributed to community empowerment ( 24 ) . the communication strategies used to educate the public are also believed to have helped the understanding of trichinellosis and involvement of the community members in the ntpp . the second factor contributing to this success : trichinellosis is caused by an agent that can be relatively easily detected in the meat . the analytic method is simple , inexpensive and rapidly provides results . furthermore , it has proven to be reliable : leclair et al . ( 2002 ) established that the digestion and double - funnel separatory procedure enables consistent detection of larvae at the level of 0.3 larvae per gram of tongue muscle . this level of sensitivity is considered sufficient to ensure that the walrus meat is safe for consumption ( 18 ) . timeliness of the communication of the results to the targeted population is an important aspect of an efficient prevention program . as walruses are large animals , one single analysis is enough to know the status of a large amount of meat . furthermore , the analyses are concentrated between july and october and these animals are harvested in limited numbers by a few captains and their crews in the same period . this program might have been more challenging to set up and maintain for small animal species that would be hunted in large numbers over a large part of the year . the timeline of the development and implementation of the ntpp and the bidirectional interweaving between science and action involved in this exercise are schematized in fig . 3 . timeline of the development and implementation of the ntpp involving interwoven science and action . the scientific characterization of the first outbreaks was essential in the development of a pilot program that evolved into the current ntpp . the new scientific knowledge generated by the program has been used as a base to continuously refine the protocols . besides scientific knowledge , the feedback of the hunters has been recorded through annual surveys , and relevant suggestions have been implemented . although successful , the ntpp had its shortcomings at the beginning of the project . firstly , the program fully relies on hunters for proper sampling and identification of each animal . sampling problems occurred in the beginning of the extension of the ntpp , making some of the samples unfit for determination of trichinella status . these difficulties were resolved by enhanced communication with the hunters and by the modification of the sampling protocol to reduce the number of needed samples per animal . tags , used to identify walrus meat , are sometimes unavailable or lost , which makes tracking back infected meat pieces complicated . this problem with the tag availability is overcome by experienced hunters , who will use handmade marks . luckily , so far the occasional failures in traceability have not hampered the elimination of infected meat . secondly , compliance with the prescribed quarantine for the meat until results of analyses are known might not be optimal . at the beginning of the program , in 1997 , several people ate improperly cooked or raw meat from an infected walrus during the quarantine period . discussion with community members suggests that compliance with the quarantine has been improved since then , although this most likely remains variable between communities ( simard , personal observation ) . some boat captains leave the meat on the boat and anchor it away from the community until results are known . in some cases the meat is quarantined in a locked building or a household freezer or shack . in other cases , community members gather when boats return from a hunt and take pieces of walrus meat that they consume , presumably cooked , prior to the release of the quarantine . unfortunately , no reliable data are available regarding people 's perception of and compliance with the ban period on freshly landed meat . the absence of recent outbreaks is indirect evidence that the ntpp is well understood and accepted by the communities ; however , more precise insight on inuit perception of the ntpp would help document and improve how people deal with meat during the quarantine period . promptness of the communication of the results is an important aspect of an efficient prevention program . the time elapsed between the landing of a walrus and the release of the results is a limitation of the ntpp . on - site analysis is not feasible because of difficulties to maintain the equipment and keep ongoing expertise in multiple local testing centres . transport of samples to established parasitology laboratories in the south was done in the 1980s but led to unacceptably long delays and did not favour implication of the population concerned in the process . consequently , the centralization of the analyses in a regional laboratory in kuujjuaq appears to be the optimal compromise , even if aerial transport of samples from local communities is still highly dependent on weather conditions . to minimize delays , samples are processed and results communicated within 24 hours of reception ( 6 ) . the different stakeholders involved in the ntpp feel strongly that the program should be perpetuated . as suggested above , there is a need to better evaluate compliance with quarantine and traceability and come up with measures to further limit the risks inherent to the quarantine period . increasing our comprehension of the epidemiology and geographic distribution of t. nativa in walrus and marine wildlife may also help to improve the prevention of this disease in the arctic and should therefore be supported . further studies on other regional sources of the parasite such as polar bear , black bear , wolf and fox meat are currently being conducted . the further prevention of trichinellosis may require either the testing of the new species involved in trichinellosis incidents or additional education . two incidents linked to bear meat have indeed been identified in tourists ( 25,26 ) . direct implication of the inuit in the scientific process of the ntpp and other health initiatives should be increased . this will greatly contribute to the appropriation of these programs by nunavimmiut and will hopefully increase the interest of inuit students in the scientific and health professions . the need to be involved in wildlife research has been expressed by native people in other regions ( 27 ) . the authors consider that the ntpp can be used as a model for a successful health - related prevention program in the arctic . the success of the program relies on many facilitating factors such as the nature of the disease and its source , the existence of an efficient analytic method , the strong involvement of the different partners including direct resource users , as well as the comprehensive bidirectional science - to - action approach that has been followed . the 2 main goals of the ntpp are to prevent human trichinellosis and to promote the walrus hunt . as shown in table i , no locally acquired walrus - linked trichinellosis outbreaks have occurred in nunavik since 1997 , even though infected walruses have been detected almost yearly since then ( fig . even if human exposures occurred in that event , the results of the analyses performed under the ntpp were communicated to the regional health services and were instrumental in the treatment of these consumers before the onset of clinical signs . ( 2002 ) , is the first report of a successful trichinellosis secondary - prevention initiative in the arctic ( 3 ) . only 2 other incidents of walrus - linked trichinellosis were recorded since the implementation of the ntpp , both during the period of generalization of the program to all walrus - hunting communities . the rare occurrence of walrus - linked trichinellosis since 1992 contrasts with the situation observed in the 1980s where outbreaks occurred every other year or so on average . in addition , it was noticed that walruses from some locations seemed to be more frequently infected . this observation is supported by the apparent decrease in prevalence associated with the avoidance of those locations by hunters . chronology and characteristics of individual trichinellosis incidents in nunavik from 1982 to 2009 epid : based on epidemiologic investigation ; lab : based on laboratory testing . the walrus involved in this outbreak was harvested and consumed in nunavut , outside the nunavik territory , and was not tested at the nrc prior to consumption . these inter - year changes are probably driven by variations in the availability of boats and financial resources , as well as weather conditions . due to this high annual variability , it is difficult to assess the impact of the ntpp on the walrus - hunting effort , even though the number of walruses harvested in nunavik appears to have stabilized over the last 10 years after an apparent decrease . nevertheless , in general , field workers and hunters feel that the ntpp has favoured the perpetuation of the hunt . the total cost of the program is difficult to evaluate since most of it is supported by in - kind participation of the different partners . nevertheless , because of the very high human and financial costs associated with the management of a single outbreak of trichinellosis in this remote and isolated arctic territory , the investment in the ntpp is considered economically sound . the ntpp can thus be considered as a successful and rational program , based on the absence of recent cases of trichinellosis acquired from walrus meat and the positive effect of the program on the continuation of the hunt . the motivation and empowerment of participating communities constitute another positive spin - off of the ntpp . first , the ntpp participates in the public mission of ensuring health in diverse ways : directly by trichinellosis prevention and indirectly because increasing food diversity is considered as a component of nutritional - diseases prevention ( 2,21 ) . the program also has positive impacts on the economic , social and cultural role of the hunt , especially because it values hunters as skilful food providers . this convergence of interest , shared by the inuit community members , the local and regional organizations and the public health authorities , is probably a major asset as regards the perpetuation of the ntpp ( 22 ) . in addition , it is recognized that effective community participation , which involves the sharing of responsibilities , contributes to the achievement of sustainable health objectives and could lead to the appropriation of research outcomes by the community ( 23,24 ) . the fact that the technical operations of the program are supported by an inuit entity ( makivik corporation ) with trained personnel ( wildlife specialists and laboratory technician ) actually facilitated communications and may have contributed to community empowerment ( 24 ) . the communication strategies used to educate the public are also believed to have helped the understanding of trichinellosis and involvement of the community members in the ntpp . the second factor contributing to this success : trichinellosis is caused by an agent that can be relatively easily detected in the meat . the analytic method is simple , inexpensive and rapidly provides results . furthermore , it has proven to be reliable : leclair et al . ( 2002 ) established that the digestion and double - funnel separatory procedure enables consistent detection of larvae at the level of 0.3 larvae per gram of tongue muscle . this level of sensitivity is considered sufficient to ensure that the walrus meat is safe for consumption ( 18 ) . timeliness of the communication of the results to the targeted population is an important aspect of an efficient prevention program . as walruses are large animals , one single analysis is enough to know the status of a large amount of meat . furthermore , the analyses are concentrated between july and october and these animals are harvested in limited numbers by a few captains and their crews in the same period . this program might have been more challenging to set up and maintain for small animal species that would be hunted in large numbers over a large part of the year . the timeline of the development and implementation of the ntpp and the bidirectional interweaving between science and action involved in this exercise are schematized in fig . 3 . timeline of the development and implementation of the ntpp involving interwoven science and action . the scientific characterization of the first outbreaks was essential in the development of a pilot program that evolved into the current ntpp . the new scientific knowledge generated by the program has been used as a base to continuously refine the protocols . besides scientific knowledge , the feedback of the hunters has been recorded through annual surveys , and relevant suggestions have been implemented . firstly , the program fully relies on hunters for proper sampling and identification of each animal . sampling problems occurred in the beginning of the extension of the ntpp , making some of the samples unfit for determination of trichinella status . these difficulties were resolved by enhanced communication with the hunters and by the modification of the sampling protocol to reduce the number of needed samples per animal . tags , used to identify walrus meat , are sometimes unavailable or lost , which makes tracking back infected meat pieces complicated . this problem with the tag availability is overcome by experienced hunters , who will use handmade marks . luckily , so far the occasional failures in traceability have not hampered the elimination of infected meat . secondly , compliance with the prescribed quarantine for the meat until results of analyses are known might not be optimal . at the beginning of the program , in 1997 , several people ate improperly cooked or raw meat from an infected walrus during the quarantine period . discussion with community members suggests that compliance with the quarantine has been improved since then , although this most likely remains variable between communities ( simard , personal observation ) . some boat captains leave the meat on the boat and anchor it away from the community until results are known . in some cases the meat is quarantined in a locked building or a household freezer or shack . in other cases , community members gather when boats return from a hunt and take pieces of walrus meat that they consume , presumably cooked , prior to the release of the quarantine . unfortunately , no reliable data are available regarding people 's perception of and compliance with the ban period on freshly landed meat . the absence of recent outbreaks is indirect evidence that the ntpp is well understood and accepted by the communities ; however , more precise insight on inuit perception of the ntpp would help document and improve how people deal with meat during the quarantine period . promptness of the communication of the results is an important aspect of an efficient prevention program . the time elapsed between the landing of a walrus and the release of the results is a limitation of the ntpp . on - site analysis is not feasible because of difficulties to maintain the equipment and keep ongoing expertise in multiple local testing centres . transport of samples to established parasitology laboratories in the south was done in the 1980s but led to unacceptably long delays and did not favour implication of the population concerned in the process . consequently , the centralization of the analyses in a regional laboratory in kuujjuaq appears to be the optimal compromise , even if aerial transport of samples from local communities is still highly dependent on weather conditions . to minimize delays , the different stakeholders involved in the ntpp feel strongly that the program should be perpetuated . as suggested above , there is a need to better evaluate compliance with quarantine and traceability and come up with measures to further limit the risks inherent to the quarantine period . increasing our comprehension of the epidemiology and geographic distribution of t. nativa in walrus and marine wildlife may also help to improve the prevention of this disease in the arctic and should therefore be supported . further studies on other regional sources of the parasite such as polar bear , black bear , wolf and fox meat are currently being conducted . the further prevention of trichinellosis may require either the testing of the new species involved in trichinellosis incidents or additional education . two incidents linked to bear meat have indeed been identified in tourists ( 25,26 ) . direct implication of the inuit in the scientific process of the ntpp and other health initiatives should be increased . this will greatly contribute to the appropriation of these programs by nunavimmiut and will hopefully increase the interest of inuit students in the scientific and health professions . the need to be involved in wildlife research has been expressed by native people in other regions ( 27 ) . the authors consider that the ntpp can be used as a model for a successful health - related prevention program in the arctic . the success of the program relies on many facilitating factors such as the nature of the disease and its source , the existence of an efficient analytic method , the strong involvement of the different partners including direct resource users , as well as the comprehensive bidirectional science - to - action approach that has been followed . the authors have not received any funding or benefits from industry or elsewhere to conduct this study
objectivesduring the 1980s , walrus - meat consumption caused infections with the parasite trichinella nativa in nunavik inhabitants . in response to these events , stakeholders set up the community - based nunavik trichinellosis prevention program ( ntpp ) . the objectives of the present communication are to review the ntpp , describe how science and action were interwoven in its development and identify its assets and limitations.study designdescriptive study.methodsthe ntpp relies on a pooled digestion assay of tongue samples taken from each harvested walrus . the public health recommendations depend on the results of the analyses : infected walrus meat should be destroyed ; parasite - free meat may be eaten raw or cooked.resultsall communities involved in the walrus hunt participate in the ntpp and a high percentage of harvested walruses are included in the ntpp . infected animals account for 2.9% of the walruses tested ( 20/694 ) since 1992 . the ntpp permitted the early management of a trichinellosis event in 1997 . since then , it prevented the new occurrence of outbreaks related to walruses hunted by nunavimmiut.conclusionsthe absence of recent major outbreaks of trichinellosis in nunavik may reasonably be attributed to the ntpp . the success of the program stands on many facilitating factors such as the nature of the disease and its source , the existence of an efficient analytic method , the strong involvement of the different partners including direct resource users , as well as the comprehensive bidirectional science - to - action approach that has been followed .
You are an expert at summarizing long articles. Proceed to summarize the following text: primary myelofibrosis ( pm ) is a myeloproliferative disorder characterized by unexplained fibrosis in the bone marrow and osteosclerosis , extramedullary hematopoiesis with marked splenomegaly , and a leukoerythroblastic anemia . other manifestations include abdominal fullness associated with the mass effect of splenomegaly , esophageal varices , and ascites caused by portal hypertension in 210% of patients 1,2 . we describe an unusual case of myelofibrosis in a splenectomized patient presenting with massive ascites , and peritoneal extramedullary hematopoiesis ; that developed nontuberculous mycobacterium infection . an 70-year - old moroccan man was admitted to our hospital with fever , abdominal fullness , and general fatigue . he had otherwise been well until 23 months before entry , when he began to note fever , poor appetite , and progressive painful abdominal fullness . because his symptoms responded poorly to therapy he decided to ask for a second opinion . on physical examination , he appeared pale and cachectic . a distended abdomen with an obvious fluid wave was noted . initial laboratory data were as follows : white - cell count 3.6 10/l ; hemoglobin 10.1 g / dl , platelet count 108 10/l . the results of all liver function test , including bilirubin , serum aspartate aminotransferase , serum alanine aminotransferase , alkaline phosphatase , and ammonium were within normal limits . serologic tests for hepatitis b surface antigen , hepatitis c antibody , and hiv elisa were negative . diagnostic paracentesis yielded yellow - colored ascites that contained 824 cells / mm , with 70% lymphocytes . the serum protein was 3.5 g / dl , and adenosine - deaminase was 9.6 a computed tomographic ( ct ) scan of the abdomen showed massive ascites , hepatomegaly , with no evidence of the thrombosis of the portal vein , and nodular thickening and enhancement of the peritoneum ( fig.1 ) . a right lower lobe consolidation on chest ct scan was observed . although the lavage fluid results were unrevealing , smear and cultures for bacteria , nocardia / actinomyces , and fungi were all negative . mycobacterium culture was positive for mycobacterium avium complex ( mac ) in bronchoalveolar lavage and urine . ct scan ( sagital and coronal views ) : nodular thickening and enhancement of the peritoneum . a biopsy specimen of the bone marrow showed decreased hematopoietic cells , fibrosis , and thickening and distortion of the bony trabeculae ( osteosclerosis ) . cd13 + , cd133+low , cd34 + , hla - dr+ , cd5+low , cd38 + , cd117low , cd71+low ) compatible with a chronic myeloproliferative neoplasm and transformation to acute myeloid leukemia . we administered hydroxyurea and corticosteroid to reduce extramedullary hematopoiesis , but he was refractory to the treatment . he received treatment with azithromycin , rifampicin , ethambutol , and decided to go back to his country where he was admitted to a hospital . extramedullary hematopoiesis refers to the production of blood cells outside the bone marrow and is a compensatory mechanism for bone marrow dysfunction . because the bone marrow reserve does not necessarily correlate with its metastatic potential , a presentation resembling metastasis of unknown origin in an otherwise asymptomatic patient with pm is possible 5 . patients with symptomatic massive ascites caused by peritoneal extramedullary hematopoiesis have been reported previously , but are limited in number 510 . symptomatic ascites in pm is rare and is often attributed to portal hypertension accompanied by ectopic hematopoiesis in the liver and spleen , and in some cases thrombosis of the portal vein 11 . in the previously reported cases , ascites occurred after splenectomy , as initial manifestation , or from 2 months to 17 years after the diagnosis of myelofibrosis was established 1,5,9 . we have not found more than nine case reports in medline ( see table1 ) . case reports of ascitis related to emh on peritoneum confirmed by peritoneal biopsy emh , extramedullary hematopoiesis . peritoneal biospy is the gold standard method as a diagonostic procedure for the peritoneal hematopoiesis , but additional cytogenetic analysis of the cells in the ascites might be useful . megakaryocytes are rarely found in peritoneal fluid and it seems to be suggestive of peritoneal extramedullary hematopoiesis 8 . there is a report about the use of busulfan and hydroxyurea for treating ascites 9 . tips may be considered as rescue management for refractory ascites secondary to portal hypertension 17 . leukemic transformation occurs in approximately 20% of patients during the first 10 years after the onset of the disease , as our case 18 . nontuberculous mycobacterial infection can cause four clinical syndromes : pulmonary disease , superficial lymphadenitis , skin / soft tissue infection , and disseminated disease in severely immunocompromised patients like our case . disseminated mac disease may complicate mac pulmonary disease through local multiplication and entry into the bloodstream with seeding of other organs and tissues 19 . isolation in urine culture is rare and should be specifically collected in special media , different from the media used in standard bacterial blood cultures . in a series of 15 patients with genitourinary infections by nontuberculous mycobacteria , only two patients had disseminated infection not related to hematologic malignancy 20 . to the best of our knowledge , tuberculous peritonitis and extramedullary peritoneal hematopoiesis has been reported previously 21 . the treatment regimen for m. avium complex pulmonary and disseminated infection includes simultaneous use of three oral antibiotics : a macrolide ( clarithromycin or azithromycin ) , ethambutol , and a rifamycin ( rifampin or rifabutin ) , for at least 1 year 22,23 . monotherapy is never advised in the treatment of mac because of the concern for developing resistance . successful treatment of disseminated mac relies on recovery of the immune system , in case of hiv patients 24,25 . this case demonstrates that refractory ascites caused by peritoneal implants of myeloid tissues may be a manifestation of myelofibrosis . nontuberculous mycobacterial disseminated infection should be included in the differential diagnosis of fever in pm patients .
key clinical messagea 70-year - old patient having massive refractory ascites in the course of idiopathic myelofibrosis was diagnosed of peritoneal extramedullary hematopoiesis and developed an overwhelming nontuberculous mycobacterial infection . the case describes this unusual infection and highlights the need for additional studies to confirm the etiology of ascites in primary myelofibrosis .
You are an expert at summarizing long articles. Proceed to summarize the following text: gene expression is regulated in chromatin through sequential recruitment of individual proteins and large multiprotein complexes to gene regulatory regions . these proteins and complexes can then function as coactivators and corepressors to aid or repress gene expression , respectively . specificity is achieved by sequence specific transcription factors that bind to consensus dna sequences found within gene regulatory regions . transcriptional coactivators often possess chromatin modifying activities that promote accessibility of the underlying dna sequences and facilitate assembly of the transcription apparatus . saga is comprised of approximately 20 subunits and bears 2 enzymes the gcn5 acetyltransferase and the non - stop deubiquitinase . combined deletion and proteomic analysis performed in saccharomyces cerevisiae has revealed that the complex is arranged in a modular fashion . the 4 modules are comprised of functionally related subunits with the spt module interacting with transcription factors , the taf module interacting with the basal transcription apparatus , and the acetyltransferase and deubiquitinase modules serving to modify chromatin and non - chromatin substrates . here we will focus on the deubiquitinase module , which is comprised of the non - stop deubiquitinase , eny2 , sgf11 , and ataxin-7 subunits . the amino ( n)-terminus of ataxin-7 extends into the deubiquitinase module , while the carboxy ( c)-terminus extends into the larger saga complex and anchors the deubiquitinase module to the complex . in humans , intergenerational expansion of cag repeats in the ataxin-7 gene results in expanded polyglutamine stretches in the n - terminus of the ataxin-7 protein . this expansion leads to spinocerebellar ataxia type 7 ( sca7 ) disease , which is characterized by progressive degeneration of the brain and the retina . the wild - type protein has 10 cag trinucleotides , and disease symptoms are observed when the repeat length extends beyond 34 , although repeat lengths greater than 400 have been reported . as repeat length increases , the age at which symptoms are first diagnosed is reduced , and the severity of the symptoms is increased . nearly 2 decades ago , our group purified the saga chromatin modifying complex from the yeast saccharomyces cerevisiae . since then , results from our group and many others have shed light on the principles regulating saga 's ability to coactivate gene expression . increasingly , however , evidence has indicated that regulation of saga in multicellular organisms involves more sophisticated mechanisms than in their unicellular counterparts . in order to gain more insight into saga function in multicellular eukaryotes this model provided powerful genetic and biochemical tools in which to explore principles underlying saga - mediated gene regulation . at that time , the identities of the subunits comprising the drosophila saga complex were largely unknown . to characterize this complex biochemically , we used cell lines stably expressing known saga subunits fused to dual flag and ha epitope tags . the complex was purified through these tagged subunits and shotgun proteomics used to determine its composition . . in silico analysis indicated that this protein shared sequence similarity with human ataxin-7 , known as sgf73 in yeast . closer analysis of the amino acid sequence of the cg9866 protein revealed conservation of a proposed the presence of this conserved sequence , suggested that we had indeed identified drosophila ataxin-7 and that it may be a subunit of the saga complex . since human and yeast ataxin-7 were found to be bona fide members of saga , we performed a series of biochemical studies to demonstrate that the putative drosophila ataxin-7 is also a component of saga . reciprocal purification of ataxin-7 followed by multidimensional protein identification technology ( mudpit ) analysis showed that it was able to co - purify saga . to establish that ataxin-7 is a stable component of the complex and not simply a strongly interacting protein , we separated putative protein complexes by size exclusion chromatography . this analysis revealed that nearly all of the ataxin-7 co - fractionated with saga , providing strong support for the conclusion that ataxin-7 is a stable component of the drosophila saga complex . moreover , the observation that virtually all ataxin-7 protein migrated with saga suggested that this is its primary function . the modular model of saga organization predicted that ataxin-7 would anchor the deubiquitinase module to the larger saga complex . this model predicts that , in the absence of ataxin-7 , the module would be released from the larger complex . to test the validity of this hypothesis , we performed gel filtration chromatography to measure the apparent sizes of wild - type saga versus saga from flies lacking ataxin-7 . we found that saga from ataxin-7-mutant flies does indeed have a reduced mass compared to that of its wild - type counterpart . immunoprecipitation using an antibody against the saga deubiquitinase , non - stop , showed that non - stop is stably associated with saga in wild - type 3rd instar larvae , but not in ataxin-7 mutant larvae . thus , the deubiquitinase module is separated from the remainder of the saga complex in ataxin-7 mutants , . biochemical and structural analysis of the yeast deubiquitinase module , considered to include the yeast orthologs of ataxin-7 , eny2 , sgf11 , and non - stop , showed that the deubiquitinase is held in an enzymatically active conformation by the presence of all members of the module . surprisingly , deubiquitinase assays revealed that the deubiquitinase module purified in the absence of ataxin-7 , and therefore separated from saga , remains enzymatically active . to confirm that the non - stop deubiquitinase module could remain enzymatically active in the absence of ataxin-7 , we reconstituted this module using purified recombinant proteins . the above finding showed that the released non - stop - containing deubiquitinase module is enzymatically active when purified from ataxin-7 mutants and when reconstituted in vitro . however , these studies did not provide insight into the behavior and localization of this module in cells lacking ataxin-7 . we took advantage of polytene chromosome spreads from the nuclei of third instar salivary glands to show that the deubiquitinase module still associated with chromatin . indeed , analysis of bulk histones acid extracted from mutant third instar larvae showed reduced levels of h2bub . under standard culture conditions , ataxin-7 homozygous mutants die pre - pupation , at the late 3rd instar stage of development . in light of the above molecular and biochemical results , we hypothesized that this lethality was due to the action of the overactive deubiquitinase . if this theory is correct , one might predict that reducing the level of the deubiquitinase would rescue loss of ataxin-7 . indeed , genetically reducing the level of non - stop in an ataxin-7 mutant fly by introducing one copy of a mutant allele suppressed the ataxin-7 lethality and allowed some mutants to survive to adulthood . this result strongly suggests that loss of ataxin-7 results in a toxic gain of activity of the non - stop deubiquitinase . since these findings deviated from predictions made from studies performed in yeast , we looked to human tissue culture models to determine whether our results were representative of saga function in multicellular organisms . in human hela cells , as in drosophila , we found a reduction in h2bub upon knockdown of ataxin-7 . moreover , this effect can be rescued by inactivating the human deubiquitinase usp22 through knock - down of the deubiquitinase module subunit sgf11 . thus , while required in yeast , the presence of ataxin-7 is dispensable for deubiquitinase module activity in higher eukaryotes . considering the phenotype associated with polyglutamine expansion of ataxin-7 , we examined the phenotypic consequences of ataxin-7 loss in flies . although the majority of ataxin-7 homozygous mutants die in the third instar larval stage of development , adult escapers can be obtained through careful culture conditions . upon analyzing these escapers , we noted that these mutant flies exhibited neural and retinal degeneration , reduced mobility , and shortened life span . importantly , this phenotype is strikingly similar to that found upon exogenous expression of the polyglutamine - expanded human ataxin-7 in drosophila . polyglutamine expansion as a cause of neurodegenerative disease was uncovered over 20 y ago when sca17 was shown to be caused by polyglutamine expansion of in the androgen receptor ( ar ) . since then , 9 genes have been identified to be subject to cag expansion , causing 9 distinct diseases . incredibly , the functions of many of these genes are only partially known , or completely unknown , and effective therapeutics for treatment of polyglutamine expansion diseases are not currently available . in order to proceed toward new discoveries , we must first determine which pathways mediate progression of these neurodegenerative diseases , and then identify novel components for intervention . we believe that drosophila models will be fruitful in the search for mechanisms contributing to neurodegeneration . for example , the gitler and bonini groups have discovered modifiers of the amyotrophic lateral sclerosis ( als)-associated 43 kda tar dna binding protein ( tdp-43 ) using a combination of yeast and drosophila models . they found that ataxin-2 , the polyglutamine - expanded protein associated with sca2 disease , strongly enhanced the toxicity of tdp-43 . the ataxin-2 and tdp-43 proteins , along with rna , exist in a complex that is mislocalized in both sca2 and als . this surprising result was the first to suggest a link between polyglutamine disease and risk for als . this potential link prompted examination of als patient samples for the prevalence of polyglutamine expanded ataxin-2 , which showed that intermediate polyglutamine expansion of ataxin-2 was present in nearly 5% of als cases . the next best predictor of als is mutant superoxide dismutase ( sod ) , which occurs in about 2% of als cases . thus , insights from a drosophila model represented a leap forward in our understanding of the pathways that contribute to als and polyglutamine disease . to further illustrate that point , these groups went on to use drosophila models to discover small molecule inhibitors suitable for modulating tdp-43-associated toxicity . the orr , westbrook , botas , and zoghbi laboratories combined efforts in a strategy that utilized drosophila and cell - based screens to identify proteins that modulate the levels of neurodegenerative disease - driving proteins . this approach proved to be powerful in identifying modifiers of polyq expanded ataxin-1 , which causes sca1disease . they chose to focus on mutants which led to a reduced level of polyq ataxin-1 protein because this also led to reduction in disease severity . this strategy was very effective , and they were able to show that down regulation of ras - mapk - msk1 pathway components results in reduced levels of ataxin-1 . importantly , pharmacological inhibitors of this pathway also reduced neurodegeneration in drosophila and in mice , raising the possibility that these inhibitors might also be effective for treatment of patients . in our study we have identified a novel , conserved , function for ataxin-7 in regulating h2b ubiquitination levels . these observations , along with previous findings by our group and others , suggest that the saga complex is an important regulator of neural development . mutants of the drosophila saga subunits non - stop , sgf11 , and ada2b all result in aberrant targeting of neural connections from the retina to the optic lobe of the brain . gcn5 mutant mice exhibit decreased brain size , severe cranial neural tube closure defects and exencephaly . now , we have provided evidence that saga complex member ataxin-7 is also necessary for regulating h2b ubiquitination and maintenance of neural stability . it is tempting to predict that polyglutamine expansion of ataxin-7 will affect h2b ubiquitination , and that removal of this histone mark might be linked directly to neurodegeneration . moving forward , d. melanogaster will certainly continue to be a driving force in the discovery of mechanisms contributing to neurodegenerative disease . to our knowledge , drosophila orthologs for 7 out of the 9 genes subject to polyq expansion have now been identified . of these thus , expanded use of drosophila to uncover novel therapeutic entry points for treatment of neurodegenerative disease is both warranted and promising . there are 9 human genes which undergo cag expansion , resulting in polyq - expansion in their protein products . here , to the best of our knowledge , we list the drosophila orthologs of the human proteins . we also note whether the effect of polyq - expansion of the native drosophila protein has been examined . 2011 ; zhai et al . 2008 ; lessing and bonini 2008 ; gu et al . 2010polyglutamine expansion diseasesdisease nameproducts of expanded gene - humandrosophila orthologreferencestudies performed with expanded drosophila ortholog*referencehuntington disease ( hd)huntingtincg9995 , htt , dhttzala et al . ( sbma)/kennedy 's diseaseandrogen receptorunknownnodentatorubral - pallidoluysian atrophyatrophin 1cg6964 , gug , grunge , atro , atrophin , l(3)03928napoletano et al . 2011yesnisoli et al . 2010sca1ataxin-1 , alt - atxn1cg4547 , atx-1 , datx-1zhai et al . 2008nosca2 and amyotrophic lateral sclerosis ( als)ataxin-2cg5166 , atx2 , ataxin-2 , datx2 , sca2 , dsca2 , l(3)06490lessing and bonini 2008nosca3 , machado - joseph diseaseataxin-3unknownnosca6a1a voltage - dependent calcium channel subunit , and a1act trascription factorcg43368 , cac , cacophony , dmca1a , nba , cg1522 , l(1)l13 , 13 , cg15928 , cac1gu et al . 2009nosca7ataxin-7cg9866 , ataxin-7 , atxn7mohan et al . 2014nosca17tata box binding protein ( tbp)cg9874 , tbp , dtbp , tfiid , tfiid , dtfiidhoey et al . there are 9 human genes which undergo cag expansion , resulting in polyq - expansion in their protein products . here , to the best of our knowledge , we list the drosophila orthologs of the human proteins . we also note whether the effect of polyq - expansion of the native drosophila protein has been examined . 2011 ; zhai et al . 2008 ; lessing and bonini 2008 ; gu et al . 2009 ; mohan et al . 2014 nearly 2 decades ago , our group purified the saga chromatin modifying complex from the yeast saccharomyces cerevisiae . since then , results from our group and many others have shed light on the principles regulating saga 's ability to coactivate gene expression . increasingly , however , evidence has indicated that regulation of saga in multicellular organisms involves more sophisticated mechanisms than in their unicellular counterparts . in order to gain more insight into saga function in multicellular eukaryotes this model provided powerful genetic and biochemical tools in which to explore principles underlying saga - mediated gene regulation . at that time , the identities of the subunits comprising the drosophila saga complex were largely unknown . to characterize this complex biochemically , we used cell lines stably expressing known saga subunits fused to dual flag and ha epitope tags . the complex was purified through these tagged subunits and shotgun proteomics used to determine its composition . . in silico analysis indicated that this protein shared sequence similarity with human ataxin-7 , known as sgf73 in yeast . closer analysis of the amino acid sequence of the cg9866 protein revealed conservation of a proposed the presence of this conserved sequence , suggested that we had indeed identified drosophila ataxin-7 and that it may be a subunit of the saga complex . since human and yeast ataxin-7 were found to be bona fide members of saga , we performed a series of biochemical studies to demonstrate that the putative drosophila ataxin-7 is also a component of saga . reciprocal purification of ataxin-7 followed by multidimensional protein identification technology ( mudpit ) analysis showed that it was able to co - purify saga . to establish that ataxin-7 is a stable component of the complex and not simply a strongly interacting protein this analysis revealed that nearly all of the ataxin-7 co - fractionated with saga , providing strong support for the conclusion that ataxin-7 is a stable component of the drosophila saga complex . moreover , the observation that virtually all ataxin-7 protein migrated with saga suggested that this is its primary function . the modular model of saga organization predicted that ataxin-7 would anchor the deubiquitinase module to the larger saga complex . this model predicts that , in the absence of ataxin-7 , the module would be released from the larger complex . to test the validity of this hypothesis , we performed gel filtration chromatography to measure the apparent sizes of wild - type saga versus saga from flies lacking ataxin-7 . we found that saga from ataxin-7-mutant flies does indeed have a reduced mass compared to that of its wild - type counterpart . immunoprecipitation using an antibody against the saga deubiquitinase , non - stop , showed that non - stop is stably associated with saga in wild - type 3rd instar larvae , but not in ataxin-7 mutant larvae . thus , the deubiquitinase module is separated from the remainder of the saga complex in ataxin-7 mutants , . biochemical and structural analysis of the yeast deubiquitinase module , considered to include the yeast orthologs of ataxin-7 , eny2 , sgf11 , and non - stop , showed that the deubiquitinase is held in an enzymatically active conformation by the presence of all members of the module . surprisingly , deubiquitinase assays revealed that the deubiquitinase module purified in the absence of ataxin-7 , and therefore separated from saga , remains enzymatically active . to confirm that the non - stop deubiquitinase module could remain enzymatically active in the absence of ataxin-7 , we reconstituted this module using purified recombinant proteins . the above finding showed that the released non - stop - containing deubiquitinase module is enzymatically active when purified from ataxin-7 mutants and when reconstituted in vitro . however , these studies did not provide insight into the behavior and localization of this module in cells lacking ataxin-7 . we took advantage of polytene chromosome spreads from the nuclei of third instar salivary glands to show that the deubiquitinase module still associated with chromatin . indeed , analysis of bulk histones acid extracted from mutant third instar larvae showed reduced levels of h2bub . under standard culture conditions , ataxin-7 homozygous mutants die pre - pupation , at the late 3rd instar stage of development . in light of the above molecular and biochemical results , we hypothesized that this lethality was due to the action of the overactive deubiquitinase . if this theory is correct , one might predict that reducing the level of the deubiquitinase would rescue loss of ataxin-7 . indeed , genetically reducing the level of non - stop in an ataxin-7 mutant fly by introducing one copy of a mutant allele suppressed the ataxin-7 lethality and allowed some mutants to survive to adulthood . this result strongly suggests that loss of ataxin-7 results in a toxic gain of activity of the non - stop deubiquitinase . since these findings deviated from predictions made from studies performed in yeast , we looked to human tissue culture models to determine whether our results were representative of saga function in multicellular organisms . in human hela cells , as in drosophila , we found a reduction in h2bub upon knockdown of ataxin-7 . moreover , this effect can be rescued by inactivating the human deubiquitinase usp22 through knock - down of the deubiquitinase module subunit sgf11 . thus , while required in yeast , the presence of ataxin-7 is dispensable for deubiquitinase module activity in higher eukaryotes . considering the phenotype associated with polyglutamine expansion of ataxin-7 although the majority of ataxin-7 homozygous mutants die in the third instar larval stage of development , adult escapers can be obtained through careful culture conditions . upon analyzing these escapers , we noted that these mutant flies exhibited neural and retinal degeneration , reduced mobility , and shortened life span . importantly , this phenotype is strikingly similar to that found upon exogenous expression of the polyglutamine - expanded human ataxin-7 in drosophila . polyglutamine expansion as a cause of neurodegenerative disease was uncovered over 20 y ago when sca17 was shown to be caused by polyglutamine expansion of in the androgen receptor ( ar ) . since then , 9 genes have been identified to be subject to cag expansion , causing 9 distinct diseases . incredibly , the functions of many of these genes are only partially known , or completely unknown , and effective therapeutics for treatment of polyglutamine expansion diseases are not currently available . in order to proceed toward new discoveries , we must first determine which pathways mediate progression of these neurodegenerative diseases , and then identify novel components for intervention . we believe that drosophila models will be fruitful in the search for mechanisms contributing to neurodegeneration . for example , the gitler and bonini groups have discovered modifiers of the amyotrophic lateral sclerosis ( als)-associated 43 kda tar dna binding protein ( tdp-43 ) using a combination of yeast and drosophila models . they found that ataxin-2 , the polyglutamine - expanded protein associated with sca2 disease , strongly enhanced the toxicity of tdp-43 . the ataxin-2 and tdp-43 proteins , along with rna , exist in a complex that is mislocalized in both sca2 and als . this surprising result was the first to suggest a link between polyglutamine disease and risk for als . this potential link prompted examination of als patient samples for the prevalence of polyglutamine expanded ataxin-2 , which showed that intermediate polyglutamine expansion of ataxin-2 was present in nearly 5% of als cases . the next best predictor of als is mutant superoxide dismutase ( sod ) , which occurs in about 2% of als cases . thus , insights from a drosophila model represented a leap forward in our understanding of the pathways that contribute to als and polyglutamine disease . to further illustrate that point , these groups went on to use drosophila models to discover small molecule inhibitors suitable for modulating tdp-43-associated toxicity . the orr , westbrook , botas , and zoghbi laboratories combined efforts in a strategy that utilized drosophila and cell - based screens to identify proteins that modulate the levels of neurodegenerative disease - driving proteins . this approach proved to be powerful in identifying modifiers of polyq expanded ataxin-1 , which causes sca1disease . they chose to focus on mutants which led to a reduced level of polyq ataxin-1 protein because this also led to reduction in disease severity . this strategy was very effective , and they were able to show that down regulation of ras - mapk - msk1 pathway components results in reduced levels of ataxin-1 . importantly , pharmacological inhibitors of this pathway also reduced neurodegeneration in drosophila and in mice , raising the possibility that these inhibitors might also be effective for treatment of patients . in our study we have identified a novel , conserved , function for ataxin-7 in regulating h2b ubiquitination levels . these observations , along with previous findings by our group and others , suggest that the saga complex is an important regulator of neural development . mutants of the drosophila saga subunits non - stop , sgf11 , and ada2b all result in aberrant targeting of neural connections from the retina to the optic lobe of the brain . gcn5 mutant mice exhibit decreased brain size , severe cranial neural tube closure defects and exencephaly . now , we have provided evidence that saga complex member ataxin-7 is also necessary for regulating h2b ubiquitination and maintenance of neural stability . it is tempting to predict that polyglutamine expansion of ataxin-7 will affect h2b ubiquitination , and that removal of this histone mark might be linked directly to neurodegeneration . moving forward , d. melanogaster will certainly continue to be a driving force in the discovery of mechanisms contributing to neurodegenerative disease . to our knowledge , drosophila orthologs for 7 out of the 9 genes subject to polyq expansion have now been identified . of these thus , expanded use of drosophila to uncover novel therapeutic entry points for treatment of neurodegenerative disease is both warranted and promising . there are 9 human genes which undergo cag expansion , resulting in polyq - expansion in their protein products . here , to the best of our knowledge , we list the drosophila orthologs of the human proteins . we also note whether the effect of polyq - expansion of the native drosophila protein has been examined . 2011 ; zhai et al . 2008 ; lessing and bonini 2008 ; gu et al . 2010polyglutamine expansion diseasesdisease nameproducts of expanded gene - humandrosophila orthologreferencestudies performed with expanded drosophila ortholog*referencehuntington disease ( hd)huntingtincg9995 , htt , dhttzala et al . 2013nospinal and bulbar muscular atrophy ( sbma)/kennedy 's diseaseandrogen receptorunknownnodentatorubral - pallidoluysian atrophyatrophin 1cg6964 , gug , grunge , atro , atrophin , l(3)03928napoletano et al . amyotrophic lateral sclerosis ( als)ataxin-2cg5166 , atx2 , ataxin-2 , datx2 , sca2 , dsca2 , l(3)06490lessing and bonini 2008nosca3 , machado - joseph diseaseataxin-3unknownnosca6a1a voltage - dependent calcium channel subunit , and a1act trascription factorcg43368 , cac , cacophony , dmca1a , nba , cg1522 , l(1)l13 , 13 , cg15928 , cac1gu et al . 2009nosca7ataxin-7cg9866 , ataxin-7 , atxn7mohan et al . 2014nosca17tata box binding protein ( tbp)cg9874 , tbp , dtbp , tfiid , tfiid , dtfiidhoey et al . there are 9 human genes which undergo cag expansion , resulting in polyq - expansion in their protein products . here , to the best of our knowledge , we list the drosophila orthologs of the human proteins . we also note whether the effect of polyq - expansion of the native drosophila protein has been examined . 2011 ; zhai et al . 2008 ; lessing and bonini 2008 ; gu et al . 2009
the saga chromatin modifying complex functions as a transcriptional coactivator for a large number of genes , and saga dysfunction has been linked to carcinogenesis and neurodegenerative disease . the protein complex is comprised of approximately 20 subunits , arranged in a modular fashion , and includes 2 enzymatic subunits : the gcn5 acetyltransferase and the non - stop deubiquitinase . as we learn more about saga , it becomes evident that this complex functions through sophisticated mechanisms that support very precise regulation of gene expression . here we describe recent findings in which a drosophila loss - of - function model revealed novel mechanisms for regulation of saga - mediated histone h2b deubiquitination . this model also yielded novel and surprising insights into mechanisms that underlie progressive neurodegenerative disease . lastly , we comment on the utility of drosophila as a model for neurodegenerative disease through which crucial and conserved mechanisms may be revealed .
You are an expert at summarizing long articles. Proceed to summarize the following text: osteoporosis is a silent metabolic process that can potentially cause fracture , disability , and increased mortality [ 13 ] . the national osteoporosis foundation ( nof ) estimates that ten million individuals already have osteoporosis and almost 34 million have low bone mass , which places them at increased risk for developing osteoporosis . a woman 's risk of breaking her hip due to osteoporosis is equal to her risk of breast , ovarian , and uterine cancers combined . there are many lifestyle risk factors for developing bone loss and osteoporosis such as a diet with poor calcium intake , lack of exercise , tobacco use , and alcohol intake [ 1 , 2 ] . women 35 years and older with a family history of osteoporosis have almost twice the risk of developing the disease , compared to women without a family history . the nof estimated that 20% of non - hispanic caucasian and asian women aged 50 and older have osteoporosis , and 52% are estimated to have low bone mass . the nof reports that one in every two caucasian women will experience an osteoporotic fracture at some point in her lifetime . it involves repetitive turnover cycles of bone osteoclasts breaking down the bone structure , which is referred to as resorption and bone osteoblast building up the bone structure , a process known as bone remodeling . in both sexes , peak bone mass is reached within three years after linear growth stops . in women perimenopausal women are especially vulnerable to bone loss due to the fluctuating and declining estrogen levels . during the perimenopausal transition , serum estradiol levels can fall from 10% to 20% , and the level of serum estrone which is a fourfold weaker than estrogen falls to about 25% to 35% of the premenopausal level . during this time , this imbalance in bone resorption and remodeling leads to accelerated bone loss . in the first five to seven years after menopause , a woman can lose up to 20% of her bone density , and this loss can lead to osteoporosis . the progression of bone loss that leads to osteoporosis can be slowed or delayed with lifestyle changes , particularly a diet with adequate calcium and vitamin d , weight - bearing physical exercise , smoking cessation , and reduction in alcohol use [ 1 , 2 ] . medications for osteoporosis are also available to minimize bone loss and prevent fracture [ 1 , 2 ] . previous studies have shown that many women of all ages lack knowledge about osteoporosis or do not perceive themselves as being at risk for developing bone loss and osteoporosis [ 711 ] . however , there is limited published literature specifically on perimenopausal women 's knowledge , health beliefs , and self - efficacy regarding osteoporosis . it is important to examine the family history , knowledge , health beliefs , and self - efficacy of perimenopausal women and to provide them adequate education . the aims of this pilot study were to ( 1 ) determine if having a family history of osteoporosis impacts knowledge , health beliefs , and self - efficacy regarding osteoporosis among perimenopausal women ( between the ages of 42 and 52 ) compared to women without a family history of osteoporosis and ( 2 ) describe the impact an osteoporosis - specific educational intervention had on the knowledge , health beliefs , and self - efficacy for this same population . the theoretical model for this study was the expanded health belief model ( ehbm ) . the health belief model ( hbm ) is a conceptual framework used to understand health behaviors and reasons for noncompliance with recommendations for health actions [ 13 , 14 ] . the health belief model was developed in the 1950s by social psychologists working for the united states public health service ( usphs ) as a way to explain why medical screening programs were not successful [ 1416 ] . the hbm has four major components for compliance with recommended health action : perceived barriers of recommended health action , perceived benefits of recommended health action , perceived susceptibility to the disease , and perceived severity of the disease . later the model was expanded to include cues to action and motivating factors . in 1988 , self - efficacy was added as a component . the core assumptions of the self - efficacy are based on the belief that three criteria primarily affect whether or not a person will take a health - related action . these three criteria are the conviction that one has the ability to initiate the activity , maintain the activity , and persist in performing the activity in the face of obstacles . for this study , a case control design was used to compare the knowledge , health beliefs , and self - efficacy of women with a family history of osteoporosis and women without a family history of osteoporosis . a pre- and posttest design was used to examine whether knowledge , health beliefs , and self - efficacy regarding osteoporosis changed after being presented in the osteoporosis - specific educational program . family history is defined as a first - degree relative and , or a grandparent . eligible participants were english speaking women between the ages of 42 and 52 still having menstrual cycles . women were excluded if they had gone 12 or more months without a menstrual period , or had had both ovaries surgically removed , and/or they had previously been diagnosed with osteoporosis . participants were recruited from an internal medicine practice with five suburban clinics . a report generated in the electronic medical record system identified women aged 4252 years . four hundred letters were sent to potential participants inviting them to participate in the study . recruitment fliers were also placed in the waiting rooms of the clinics . some of the women who participated in the study recommended the study to their friends , which was helpful in recruiting participants . baseline demographic information regarding age , race , educational level , marital status , and family history of osteoporosis was obtained from the participants . the participants then completed three valid and reliable standardized tools : the osteoporosis knowledge test ( okt ) , the osteoporosis health belief scale ( ohbs ) , and the osteoporosis self - efficacy scale ( oses ) . permission to use these tools was granted by a coauthor of these instruments ( p. gendler , personal communication , october 27 , 2011 ) . study materials were coded , which provided the ability to compare study materials , while maintaining confidentiality of the responses . the okt ( revised 2011 ) is a 32-item multiple choice instrument that measures knowledge of osteoporosis . the okt has two subscales : okt nutrition ( items 111 and 1832 ) and okt exercise ( items 117 and 3032 ) . the okt nutrition and okt exercise subscales both share 14 common items ( items 111 and 3032 ) . , mary horan , ph.d . , and phyllis gendler , ph.d . and was revised by phyllis gendler , ph.d . , cynthia coviak , ph.d . , question 26 was developed as an addition to the osteoporosis knowledge test by pamela von hurst ( 2006 ) . the reliability coefficient for internal consistency ( kr 20 ) of the okt ( revised 2011 ) for the total scale was .839 . questions were examined for difficulty , effectiveness of distracters , and discrimination ( p. gendler , personal communication , october 27 , 2011 ) . the ohbs has 42 items and consists of two subscales : the osteoporosis health belief calcium scale ( ohbcs ) and the osteoporosis health belief exercise scale ( ohbes ) . the ohbcs and ohbes share three subscales : perceptions of osteoporosis regarding seriousness , susceptibility , and general health motivation . the subscales measuring the concepts of barriers and benefits are specific to calcium intake and exercise behavior and are different for the two scales . the participants rate each item using a 5-point scale ( 1 : strongly disagree to 5 : strongly agree ) . the range of scores for each subscale is 6 to 30 , with a possible total range of 42 to 210 . the researchers determined internal consistency of the ohbcs and ohbes by cronbach alpha reliability with coefficients ranging from .61 ( health motivation ) to .80 ( susceptibility ) . the percentages of variance explained by susceptibility , barriers , benefits , seriousness and heath motivation were 14.4 , 12.4 , 9.1 , 7.7 , and 5.8 , respectively . the percentages of variance for the ohbes accounted for susceptibility , benefits exercise , barriers exercise , seriousness , and health motivation were 15.9 , 12.1 , 9.2 , 6.4 , and 5.7 , respectively . the oses tool consists of two subscales : the ose - exercise scale and ose - calcium scale . the items are rated by the participants on their confidence about engaging in osteoporosis preventive behaviors . the researchers report that the reliability and validity of the oses were accomplished through use of a principle factor analysis . the ose - exercise and ose - calcium had internal consistency estimates of .94 and .93 , respectively . after completing the okt , ohbs , and oses , the participants were presented an educational program entitled healthy bones ; build them for life purchased from the nof . this education program is a 45-minute power point presentation that was developed by the nof in an effort to increase awareness about bone health and osteoporosis and to promote bone health practices . the participants were also given five handouts developed by the nof : who gets osteoporosis : factors that put you at risk , building bones to last a lifetime , what you should know about calcium , vitamin d and bone health , and exercise for your bone health . the participants were sent home with a postage paid envelope , void of the participants address , containing the okt , ohbs , and oese . they were asked to complete the surveys two months after attending the educational program and return them . after the surveys were returned , the participants were sent a $ 20 gift card of their choosing . initially the sample included 20 women with a family history of osteoporosis and 22 women without a family history of osteoporosis . the posttest was returned by only 37 of the women , 18 women with a family history of osteoporosis , and 19 women without a family history . the mean age for women with a family history was 4446 and 4749 both at 38.9% . the mean educational level for the women with a family history was 50% with some college / associate degree . the mean age for women without a family history of osteoporosis was 4749 and 5052 both at 32% . the education level for the women without a family history was 36% for both some college / associate degree and baccalaureate degree . eighty - six percent of the women without a family history of osteoporosis were caucasian . knowledge was analyzed by the total of knowledge questions answered correctly on the osteoporosis knowledge test . to analyze the differences between the two group 's knowledge scores , there was no statistically significant difference in the knowledge of osteoporosis on the pretest between women with a family history of osteoporosis with a mean of 12.8 questions answered correctly and women without a family history of osteoporosis with a mean of 12.2 questions answered correctly . almost all of the participants in both groups ( 95% ) knew that having a parent or grandparent with osteoporosis increases a person 's chance of developing osteoporosis . additionally , 83% of all participants knew that a diet low in dairy products , being menopausal ( 88% ) , smoking daily ( 81% ) , and having an eating disorder ( 88% ) increase a person 's risk of developing osteoporosis . however , only 16% of all the participants knew that elderly men had an increased chance of developing osteoporosis , and only 4.2% of all the women knew the amount of exercise recommended per week to strengthen bones . while 83% of all of the women knew that vitamin d is required for the absorption of calcium , only 16% knew the daily recommended amount of vitamin d required for the absorption of calcium , and only 12% knew that salmon was a good food source for vitamin d. to analyze the differences between the two group 's knowledge scores after the osteoporosis - specific education intervention , a paired t - test was calculated . the results revealed that there was a statistically significant increase in knowledge scores in both groups . for women with a family history the posttest mean was 25.8 questions answered correctly ( t = 10.214 ; df = 17 , p < .001 ) . the women without a family history had a highly significant difference in posttest scores with the mean of 26.11 questions answered correctly ( t = 21.881 ; df = 18 ; p < .001 ) . health beliefs were examined with the osteoporosis health belief scale . to analyze health belief differences on the pretest between the women with a family history of osteoporosis and the women without a family history of osteoporosis , there were a possible range of 42 to 210 for the total ohbs and a possible range of 6 to 30 for the subscales . there was a statistically significant difference with regard to perceived susceptibility to developing osteoporosis between women with a family history of osteoporosis ( mean 21.66 ) and those without a family history of osteoporosis ( mean 17.31 ) ( t = 3.403 ; df = 38.04 ; p = .032 ) . there was no statistically significant difference in perceived seriousness , benefits of exercise , benefits of calcium intake , barriers to exercise , and barriers to calcium intake between the two groups , nor was there a significant difference observed between the two groups on health motivation . to analyze the differences between the two group 's health belief 's scores after the osteoporosis - specific education intervention , a paired t - test was calculated . the results for the women with a family history of osteoporosis showed a slight increase in mean perceived susceptibility , seriousness , benefits to exercise , benefits to calcium , and health motivation . the only comparison that showed a statistical difference was in the benefits of exercise ( t = 3.072 ; df = 17 ; p = .007 ) . the women without a family history of osteoporosis had a slight increase in mean perceived susceptibility , benefits of calcium , and health motivation . the only statistically significant difference was in the benefit of calcium ( t = 5.189 ; df = 18 ; p < .001 ) . this is displayed in table 3 . when analyzing the results from the two subscales of the osteoporosis self - efficacy scale , the ose - exercise scale , and ose - calcium scale , independent t - tests were calculated . on the pretest the women with a family history of osteoporosis had a mean confidence of 60% for self - efficacy to exercise and a 78% confidence in self - efficacy for calcium consumption . the women without a family history had a mean confidence of 62% for self - efficacy to exercise and a 79% confidence in self - efficacy for calcium consumption . in analyzing the differences between the two group 's self - efficacy scores after the osteoporosis - specific education intervention the women with a family history of osteoporosis had a mean confidence of 62% for self - efficacy to exercise and an 80% confidence in self - efficacy for calcium consumption . the women without a family history of osteoporosis had a mean confidence of 67% for self - efficacy to exercise and an 83% confidence in self - efficacy for calcium consumption . although the women without a family history of osteoporosis had a higher mean confidence of self - efficacy to exercise and for calcium consumption , there were no significant findings . the aims of this pilot study were to determine if having a family history of osteoporosis impacts knowledge , health beliefs , and self - efficacy regarding osteoporosis among perimenopausal women between the ages of 42 and 52 and secondly to describe the impact an osteoporosis - specific educational intervention had on the knowledge , health beliefs , and self - efficacy for this same population . it was determined that study participants had limited knowledge of osteoporosis and that there was no difference in knowledge between women who have a family history of osteoporosis and women who do not have a family history of osteoporosis . the major finding of this study was that osteoporosis - specific education clearly increases knowledge . it was a sensible finding that women with a family history of osteoporosis felt more susceptible to developing osteoporosis than those who do not have a family history of osteoporosis , as women with a family history of osteoporosis have almost double the risk of developing osteoporosis . after the education program , perceived susceptibility did increase in both groups , although this finding was not statistically significant . the women in both groups perceived the barriers to exercise and calcium as low and the benefits of exercise and calcium as high . this finding is consistent with findings in previous work with younger women and adolescent girls [ 7 , 11 ] . it is an interesting finding in this study that the women with a family history of osteoporosis perceived exercise more beneficial and women without a family of osteoporosis perceived calcium more beneficial after the education program . the three criteria of self - efficacy are the conviction that one has the ability to initiate the activity , maintain the activity , and persist in performing the activity in the face of obstacles . it is important to note that in this study self - efficacy total mean scores for both groups of women were above average before the education program . the mean scores for both groups increased after the education program , with the greater impact in the women without a family history of osteoporosis . the study was also limited because there was limited diversity of the study population . of the study population , the major finding of this study was that the osteoporosis - specific education program clearly increased knowledge and some health beliefs in women with and without a family history of osteoporosis . perimenopausal women are especially vulnerable to bone loss due to the fluctuating and declining levels of estrogen that are needed to maintain bone health . conversation and education about the risk factors and preventative measures of osteoporosis need to start early in the perimenopausal years and not be delayed to the postmenopausal years as this may reduce morbidity later in life .
the aims of this pilot study were to ( 1 ) determine if having a family history of osteoporosis impacts knowledge , health beliefs , and self - efficacy regarding osteoporosis among perimenopausal women aged 4252 and to ( 2 ) describe the impact of an osteoporosis - specific educational intervention had on the knowledge , health beliefs , and self - efficacy of this population . participants completed three surveys measuring knowledge , health beliefs , and self - efficacy related to osteoporosis before and two months after the educational program . at baseline , no differences were noted in knowledge of osteoporosis among women with and without a family history of osteoporosis , although women with a family history perceived a greater susceptibility for developing osteoporosis than women without the family history . findings indicate that both groups increased in knowledge of osteoporosis ( p < .001 ) . benefits of calcium increased in the women without a family history of osteoporosis ( p < .001 ) and benefits of exercise increase in women with a family history of osteoporosis ( p = .007 ) . there were no significant statistical findings regarding self - efficacy between the two groups of women . findings indicate that an osteoporosis - specific educational program improves perimenopausal women 's knowledge and some health beliefs .
You are an expert at summarizing long articles. Proceed to summarize the following text: only 10%20% of patients with hepatocellular carcinoma ( hcc ) have the option for curative therapy such as resection , transplantation , or local ablation.1 according to the barcelona clinic liver cancer tumor staging , transarterial chemoembolization ( tace ) is performed in patients with large or multinodal tumors where curative treatments are not feasible . however , these patients need to have a sufficient remaining liver function ( child - pugh class a and b liver cirrhosis ) and absence of extrahepatic tumor manifestation , macrovascular invasion , and cancer - related symptoms ( barcelona clinic liver cancer intermediate stage b).2,3 there is evidence that tace is able to improve survival in these patients compared to symptomatic treatments alone.46 like every invasive treatment , tace bears the risk of side effects . the most common adverse events are abdominal pain , nausea , fever , and elevated transaminases during the initial days post intervention . severe side effects such as hepatic decompensation , gastrointestinal bleeding , or treatment - related death are found in < 10% of the patients treated with tace.2,3 during tace , a chemotherapeutic agent is administered into the feeding artery of the hcc nodule followed by application of an embolic material to gain a high intratumoral concentration of chemotherapeutics and tumor necrosis.7 nonresorbable drug - eluting beads ( deb ) are new embolic agents used in tace . first studies showed higher intratumoral and lower systemic concentration of doxorubicin compared to the common doxorubicin lipiodol mixture , which is used as an embolic agent in conventional tace ( ctace).810 prospective randomized controlled trials comparing ctace with deb - tace showed less systemic side effects of the chemotherapeutic agent but no superiority in tumor response or overall survival after deb - tace.7,11 however , some retrospective studies showed better treatment response and longer time to progression after deb - tace.12,13 our retrospective study was conducted to compare tumor response and survival and postinterventional side effects such as pain and fever that influence the postinterventional quality of life in hcc patients treated with either ctace or deb - tace . data of 32 patients suffering from unresectable hcc and treated with tace were retrospectively analyzed . hcc was diagnosed according to the american association for the study of liver diseases practice guidelines with one or two dynamic imaging techniques . in case of doubt , a biopsy was performed.3 the protocol for this study was approved by the ethics committee of the university of wuerzburg ; as such , no written informed consent from each individual patient was required for inclusion to this study , since it was retrospective , and all data was de - identified . procedural consent for tace patient s inclusion criteria were 1 ) diagnosed hcc outside the milan criteria ( one single lesion < 5 cm or three lesions <3 cm each14 ) and 2 ) treatment with deb - tace or ctace . patients were excluded from the analysis if 1 ) the tumor was inside the milan criteria ; 2 ) the patients were consecutively treated with curative procedures ( transplantation , resection , or local ablative methods ) after initial tace ; or 3 ) the patients received selective internal radiation therapy after initial tace . as the treatment with targeted therapy like sorafenib is a common part in the treatment of advanced hcc nowadays , patients receiving this form of medication were not excluded from the study . sorafenib was administered only in case of recurrent disease not feasible for tace or as additive therapy after tace . decision for targeted therapies was made by interdisciplinary tumor board consensus . as described elsewhere , each procedure was performed via femoral artery puncture.12,15 first , standardized pressure arteriography of celiac and superior mesenteric arteries was performed to investigate arterial anatomy of the liver , vascular supply of tumor nodes , and patency of the portal vein . chemotherapeutics and embolic agents were infused either superselectively into the tumor - supplying arteries or nonselectively into a single liver lobe or the whole organ according to tumor size and location . a superselective procedure was performed in 33.3% of the patients in the ctace group and 21.4% of patients in the deb - tace group . for ctace , a mixture of doxorubicin ( n=11 ) or epirubicin ( n=7 ) and lipiodol was used . for deb - tace , the choice of chemotherapeutic agent and the decision of ctace versus deb - tace were at the discretion of the performing radiologist . for deb - tace , deb ( dc bead ; biocompatibles , farnham , uk ) with a diameter of 300500 m were used . according to the body surface area and tumor size , embolization with microspheres ( bead block ; biocompatibles ) after ctace or deb - tace application was a facultative treatment to consolidate stasis within the feeding arteries . the primary outcomes of the study were 30-day mortality , peri - interventional need of pain medication , fever 38.0c ( 100.4f ) , and elevation of transaminases to evaluate the appearance of a postembolization syndrome after ctace or deb - tace . another primary outcome of the study was overall and median survival after the initial tace . the secondary outcomes were local and extrafocal recurrence of the disease after 3 months and 6 months detected in radiologic imaging , which is defined as newly detected areas of contrast enhancement at the edge of the known hcc nodules or newly detected intra- or extrahepatic manifestation of hcc . the patients were usually followed up every 3 months after the initial tace with the same cross - sectional multiphase imaging used before the initial tace ( multislice computed tomography or dedicated magnetic resonance imaging ) . the follow - up cases and their further treatment were discussed in an interdisciplinary conference . a p - value < 0.05 was defined to be significant . a unpaired t - test was performed to investigate continuous variables , and a chi - square test was used to investigate categorical variables . data of 32 patients suffering from unresectable hcc and treated with tace were retrospectively analyzed . hcc was diagnosed according to the american association for the study of liver diseases practice guidelines with one or two dynamic imaging techniques . in case of doubt , a biopsy was performed.3 the protocol for this study was approved by the ethics committee of the university of wuerzburg ; as such , no written informed consent from each individual patient was required for inclusion to this study , since it was retrospective , and all data was de - identified . procedural consent for tace patient s inclusion criteria were 1 ) diagnosed hcc outside the milan criteria ( one single lesion < 5 cm or three lesions <3 cm each14 ) and 2 ) treatment with deb - tace or ctace . patients were excluded from the analysis if 1 ) the tumor was inside the milan criteria ; 2 ) the patients were consecutively treated with curative procedures ( transplantation , resection , or local ablative methods ) after initial tace ; or 3 ) the patients received selective internal radiation therapy after initial tace . as the treatment with targeted therapy like sorafenib is a common part in the treatment of advanced hcc nowadays , patients receiving this form of medication were not excluded from the study . sorafenib was administered only in case of recurrent disease not feasible for tace or as additive therapy after tace . as described elsewhere , each procedure was performed via femoral artery puncture.12,15 first , standardized pressure arteriography of celiac and superior mesenteric arteries was performed to investigate arterial anatomy of the liver , vascular supply of tumor nodes , and patency of the portal vein . chemotherapeutics and embolic agents were infused either superselectively into the tumor - supplying arteries or nonselectively into a single liver lobe or the whole organ according to tumor size and location . a superselective procedure was performed in 33.3% of the patients in the ctace group and 21.4% of patients in the deb - tace group . for ctace , a mixture of doxorubicin ( n=11 ) or epirubicin ( n=7 ) and lipiodol was used . for deb - tace , the choice of chemotherapeutic agent and the decision of ctace versus deb - tace were at the discretion of the performing radiologist . for deb - tace , deb ( dc bead ; biocompatibles , farnham , uk ) with a diameter of 300500 m were used . according to the body surface area and tumor size , the concentration of the used chemotherapeutic drug was determined . embolization with microspheres ( bead block ; biocompatibles ) after ctace or deb - tace application was a facultative treatment to consolidate stasis within the feeding arteries . the primary outcomes of the study were 30-day mortality , peri - interventional need of pain medication , fever 38.0c ( 100.4f ) , and elevation of transaminases to evaluate the appearance of a postembolization syndrome after ctace or deb - tace . another primary outcome of the study was overall and median survival after the initial tace . the secondary outcomes were local and extrafocal recurrence of the disease after 3 months and 6 months detected in radiologic imaging , which is defined as newly detected areas of contrast enhancement at the edge of the known hcc nodules or newly detected intra- or extrahepatic manifestation of hcc . the patients were usually followed up every 3 months after the initial tace with the same cross - sectional multiphase imaging used before the initial tace ( multislice computed tomography or dedicated magnetic resonance imaging ) . the follow - up cases and their further treatment were discussed in an interdisciplinary conference . a unpaired t - test was performed to investigate continuous variables , and a chi - square test was used to investigate categorical variables . patients characteristics are shown in table 1 . in all , 18 patients ( 56.3% ) received ctace and 14 patients ( 43.8% ) deb - tace . there were no differences due to sex , age , tumor burden , child - pugh stage , okuda stage , and initial model for end - stage liver disease score . in average , 1.61 procedures per patient in the ctace group and 1.71 procedures per patient in the deb - tace group have been performed . a total of ten patients ( 55.6% ) in the ctace group and two patients ( 14.3% ) in the deb - tace group received additional bland embolization . none of the patients received sorafenib before or during the first tace procedure . in all , 33.3% of patients in the ctace group and 57.1% of patients in the deb - tace group received sorafenib in the course after the first tace ( p=0.1780 ) . there was no statistically significant difference between ctace and deb - tace with regard to 30-day mortality . in all , three patients died within 30 days after initial tace , two in the ctace group ( 11.1% ) and one in deb - tace group ( 7.1% ) . the reasons for death were gallbladder rupture with consecutive abdominal sepsis and decompensation of liver cirrhosis in the ctace group and rupture of the tumor with consecutive hemorrhagic shock in the deb - tace group . between the ctace and deb - tace groups , no statistically significant differences could be detected with regard to mean hospital stay ( 3.7 days vs 3.0 days ) and appearance of postinterventional fever of 38.0c ( 100.4f ) within the first 3 days after intervention ( 50.0% vs 50.0% ) . in all , five patients ( 31.3% ) in the ctace group and ten patients ( 71.1% ) in the deb - tace group required postinterventional application of analgesics ( p=0.0281 ) . intravenous analgesics as well as opiates were also needed more often in patients treated with deb - tace ( table 2 ) . additionally , deb - tace patients did show a trend toward a higher increase in transaminase levels within 3 days after intervention , although differences did not reach statistical significance ( data not shown ) . figure 1 shows the overall survival after initial tace of patients included in this study . a difference or a trend toward superiority of one of the procedures in the overall survival after initial tace could not be detected . the overall median survival after the initial procedure was 10.8 months in the ctace group and 9.2 months in the deb - tace group , showing no statistically significant difference . therefore , a substratification in the following subgroups has been performed : bilobar tumor burden , multinodular tumor burden ( 4 tumor nodules ) , child - pugh class a cirrhosis , and not superselective procedure ( tace of an entire liver lobe or whole liver ) . in none of the subgroups , superiority of a single procedure in the overall or median survival after the initial tace could be detected ( table 3 ) . yet , in case of multinodular tumor burden in patients with child - pugh class a cirrhosis , a trend toward longer median survival in ctace could be detected . local or extrafo0cal recurrence of hcc 3 months and 6 months after the initial tace as defined earlier is shown in table 4 . however , there was a trend toward a higher rate of extrafocal recurrence in patients treated with deb - tace . there was no statistically significant difference between ctace and deb - tace with regard to 30-day mortality . in all , three patients died within 30 days after initial tace , two in the ctace group ( 11.1% ) and one in deb - tace group ( 7.1% ) . the reasons for death were gallbladder rupture with consecutive abdominal sepsis and decompensation of liver cirrhosis in the ctace group and rupture of the tumor with consecutive hemorrhagic shock in the deb - tace group . between the ctace and deb - tace groups , no statistically significant differences could be detected with regard to mean hospital stay ( 3.7 days vs 3.0 days ) and appearance of postinterventional fever of 38.0c ( 100.4f ) within the first 3 days after intervention ( 50.0% vs 50.0% ) . in all , five patients ( 31.3% ) in the ctace group and ten patients ( 71.1% ) in the deb - tace group required postinterventional application of analgesics ( p=0.0281 ) . intravenous analgesics as well as opiates were also needed more often in patients treated with deb - tace ( table 2 ) . additionally , deb - tace patients did show a trend toward a higher increase in transaminase levels within 3 days after intervention , although differences did not reach statistical significance ( data not shown ) . figure 1 shows the overall survival after initial tace of patients included in this study . a difference or a trend toward superiority of one of the procedures in the overall survival after initial tace could not be detected . the overall median survival after the initial procedure was 10.8 months in the ctace group and 9.2 months in the deb - tace group , showing no statistically significant difference . therefore , a substratification in the following subgroups has been performed : bilobar tumor burden , multinodular tumor burden ( 4 tumor nodules ) , child - pugh class a cirrhosis , and not superselective procedure ( tace of an entire liver lobe or whole liver ) . in none of the subgroups , superiority of a single procedure in the overall or median survival after the initial tace could be detected ( table 3 ) . yet , in case of multinodular tumor burden in patients with child - pugh class a cirrhosis , a trend toward longer median survival in ctace could be detected . local or extrafo0cal recurrence of hcc 3 months and 6 months after the initial tace as defined earlier is shown in table 4 . however , there was a trend toward a higher rate of extrafocal recurrence in patients treated with deb - tace . for patients with hcc not suitable for curative treatment options , transarterial therapy with chemotherapeutic deb is a new alternative for locoregional treatment . several preclinical and clinical studies showed high intratumoral and low systemic dosages of doxorubicin8,10,16 resulting in a satisfying safety profile and high rates of tumor response.17,18 although these studies showed promising results , prospective randomized controlled trials could not prove significant superiority of deb - tace over ctace regarding tumor response in the overall setting.7,11 however , less drug - related side effects , eg , fever , liver toxicity events , cardiotoxicity , and alopecia , after the application of deb were observed.7,15 survival rates comparing ctace and deb - tace have been already investigated by prospective randomized controlled trials , in retrospective studies as well as in meta - analyses , and the results are heterogeneous ( table 5 ) . two of these studies showed a prolonged overall and median survival in patients treated with deb - tace,12,19 while the other studies could not demonstrate relevant differences.11,13,20,21 one study even showed a higher response rate and a longer time to progression after conventional chemoembolization.22 our data did not show any differences in the overall and median survival , but there were differences in the median survival showing better results after ctace in patients with multinodular or bilobar tumor burden and in patients having liver cirrhosis in child - pugh class a. investigating local recurrence of disease after 3 months and 6 months also did not reveal any differences between the study groups . surprisingly , there was a trend toward more cases of extrafocal recurrence in the deb - tace group , although this difference was not statistically significant . this could be explained by the higher amount of multinodular hccs in the deb - tace group . as mentioned earlier it was also shown that fewer side effects occurred after deb - tace in comparison to the conventional procedure.7,15 in contrast to these data , there was no trend for less toxic effects of the deb in comparison to ctace in our analysis . this is in accordance with the data of adverse events published by the precision italia study group comparing deb - tace and ctace in 177 patients,11 showing no differences in the rate of postinterventional fever , increased alanine transaminase , nausea , or liver function worsening . however , this randomized study revealed advantage of deb - tace in postprocedural abdominal pain . in contrast to this result , our analysis showed that deb - tace - treated patients experienced increased postinterventional pain and consecutively required statistically significant more pain medication within the first 3 days after the procedure . although our data are limited by the small study population and its retrospective character , this issue was never described in previous studies comparing the two modalities of tace . to avoid , that the extend of liver tissue treated with tace of both modalities influenced these results , we performed a subgroup analysis of patients receiving tace of an entire liver lobe or the whole liver . this subgroup analysis confirmed , that patients receiving deb - tace required significantly more opiate containing pain medication ( 50.0% vs 8.3% , p=0.0289 ) . postprocedural fever was not a factor for the administration of postoperative pain medication or nonsteroidal anti - inflammatory drug since there was no difference in the appearance of postinterventional fever in the two groups . the significant difference in pain medication with a fivefold higher application rate of opiates in the deb - tace group is not explainable by the higher amount of multinodular tumor burden in the deb - tace group . the major adverse events appeared in 9.4% of patients with no advantage for ctace or deb - tace . in comparison to other study collectives however , this high 30-day mortality could be due to a high rate of not superselective procedures ( 71.9% ) in patients with a high tumor burden with consecutive higher risk of maldistribution of embolic agent or severe liver toxicity with consecutive liver failure . our study could not confirm superiority regarding tumor response and overall survival of deb - tace over ctace . surprisingly , a higher rate of postinterventional pain was detected and described for the first time after deb - tace .
purposein hepatocellular carcinoma patients with large or multinodal tumors , where curative treatment options are not feasible , transarterial therapies play a major role . transarterial chemoembolization ( tace ) with drug - eluting beads ( deb - tace ) is a promising new approach due to higher intratumoral and lower systemic concentration of the chemotherapeutic agent compared to conventional tace ( ctace).patients and methodsin a retrospective analysis , 32 patients with hepatocellular carcinoma who received either deb or a ctace were compared regarding survival time , disease recurrence , and side effects such as pain and fever.resultsno significant differences could be detected between the ctace and deb - tace groups with regard to mean hospital stay , appearance of postinterventional fever , or 30-day mortality . however , the application of intravenous analgesics as postinterventional pain medication was needed more often in patients treated with deb - tace ( 57.1% vs 12.5% , p=0.0281 ) . the overall median survival after the initial procedure was 10.8 months in the ctace group and 9.2 months in the deb - tace group , showing no significant difference.conclusionno survival benefit for patients treated with either deb - tace or ctace was observed . surprisingly , a higher rate of postinterventional pain could be detected after deb - tace .
You are an expert at summarizing long articles. Proceed to summarize the following text: parkinson 's disease ( pd ) is a neurodegenerative disorder of the central nervous system that belongs to a group of conditions referred to as movement disorders . efforts to understand the genetic basis of this disease have led to the identification of a number of gene mutations accounting for a small proportion of pd cases . although the role of mitochondria in pd has been debated inconclusively in the past few decades , it was the identification of pd - causative mutations in the mitochondrial putative protein kinase pink1 that helped reignite the interest in the potential role of this organelle in the pathophysiology of familial pd . it is now becoming clear that pinki acts in a mitochondrial quality control pathway and that other genes implicated in this disease are under its control . this suggests that a subset of genes involved in pd converge in a common pathway that modulates the fidelity of mitochondria . given that most pd cases are sporadic with no known specific cause , this raises the interesting hypothesis that impaired mitochondrial quality control might be a defining causative feature of this disease . the purpose of this review is to summarise some of the recent molecular determinants of mitochondrial quality control regulated by pink1 . these microscopic life forms reside within cells of all living things and communicate using the force . the force is an energy field created by all living things and harvested by the jedi knights to protect the galaxy . midichlorians not only help to communicate the force , but they are also symbionts with all living things . without them , life could not exist . the creator of star wars , george lucas , stated that the midichlorians are based on the endosymbiotic theory , which explores the origin of mitochondria and plastids present in eukaryotic cells . in fact , midichlorians are loosely based on mitochondria , an organelle that sustains life in eukaryotic cells of multicellular organisms ; however , these organelles are also known to bring about death and destruction within cells . it is widely accepted that the acquisition of mitochondria is a crucial event in the evolution of the eukaryotic cell . according to the endosymbiotic theory , mitochondria evolved from a metabolically driven symbiotic relationship between aerobic bacteria and a nucleus - containing cell . as descendants of the original symbiont , mitochondria are organelles highly efficient in their ability to utilise molecular oxygen ( o2 ) and organic substrates , such as glucose and pyruvate , and to produce cellular energy in the form of atp . the molecular machinery present in mitochondria for energy production is collectively referred to as the electron transport chain ( etc ) . three of the complexes ( i , iii and iv ) pump protons ( h ) outwardly across the inner membrane to establish a gradient necessary for the production of atp by a molecular motor ( complex v or atp synthase ) . during electron transport , o2 is converted to h2o , and , particularly at complexes i and ii , the free radical superoxide \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { { { \hbox{o}}_2}^ { - } } \right ) $ $ \end{document } is also generated as a by - product . two important cofactors that modulate energy and free radical production are coenzyme q10 at complex ii and cytochrome c at complex iv . it is noteworthy that excessive production of superoxide can enhance oxidative stress in dopaminergic neurons , eventually contributing to the demise of these cells in pd . mitochondria play a key role in eukaryotic cells by supplying the majority of the cellular energy in the form of atp . however , a cell death - related role for this organelle is also now clearly established . when eukaryotic cells in multicellular organisms become worn out or damaged , they undergo cellular suicide ( i.e. apoptosis ) . during this process , when this process lacks proper control , either decreased or increased cell loss can occur , leading to diseases such as cancer or neurodegeneration . apoptosis is actively controlled by mitochondria . when the mitochondrial pathway of apoptosis is activated , these organelles release several proteins that activate several proteases and other hydrolytic enzymes , leading to proteolysis , dna fragmentation and chromatin condensation . the main mitochondrial effectors of apoptosis are proteins that paradoxically play important survival roles in non - apoptotic cells . these effectors include cytochrome c , which , in normal settings , is a component of the etc , and the serine protease htra2 ( also known as omi ) which seems to play a protective role during stress in the mitochondria of healthy cells . the mitochondrial pathway of apoptosis is regulated by the bcl-2 family proteins ( reviewed in youle and strasser ) and involves the activation of bcl-2 family members , such as bax and bak , which act by forming pores in the outer mitochondrial membrane ( omm ) and allow the release of several pro - apoptotic proteins from the intermembrane space into the cytosol . the role of enhanced apoptosis as a causative mechanism for the death of dopaminergic neurons in pd patients is still controversial . however , given that healthy mitochondria contain a multitude of molecules that , when released into the cytosol , lead to apoptotic cell death , it is reasonable to assume that the compromise of mitochondrial function in dopaminergic neurons contributes to their demise through the engagement of this form of cell suicide . the majority of this energy is used for reversing the movement of ions that bring about action potentials and synaptic transmission in neurons . neurons have very low glycolytic rates ( reviewed in bolanos and almeida ) and therefore require mitochondria for energy production . mitochondria also sequester and buffer cytoplasmic calcium ; thus , their localisation plays a role in local regulation of intracellular calcium dynamics . neurons are large , specialised cells , and the biogenesis of mitochondria in neurons takes place in the soma . impressively , these organelles will travel up to 1 m in human motor neurons and , taking into account their maximum travelling speed , will take a minimum of 8 days to reach their destination . the majority of long - distance mitochondrial transport is mediated by motor proteins powered by atp hydrolysis that shuttle mitochondria along microtubules ( reviewed in macaskill and kittler ) . such mitochondrial transport is bidirectional , as damaged mitochondria may need to travel back to the cell soma for autophagic degradation . this degradation is mediated through autophagy , a molecular recycling programme for impaired and defective cellular components . in neurons , synaptic transmission requires high levels of cellular atp for numerous energy - consuming processes , including the maintenance of synaptic membrane potential and reloading of synaptic vesicles with neurotransmitters . in addition , synaptic terminals may require mitochondria for efficient sequestration of calcium ions ( ca ) that are released into the cytosol to elicit the fusion of synaptic vesicles with the plasma membrane ( reviewed in ly and verstreken ) . adult dopaminergic neurons of the substantia nigra pars compacta ( snc ) that degenerate in pd are known to generate action potentials in the absence of any synaptic input . the mechanism behind this autonomous firing capacity involves ion channels that enable the entry of ca into the cytosol ( reviewed by chan , gertler and surmeier ) . this ca is actively removed by active pumping across the plasma membrane or sequestration in the lumen of the endoplasmic reticulum ( er ) or the mitochondrial matrix , a process that requires energy in the form of atp . also , active ca buffering by mitochondria is likely to have a significant effect in the physiology of this organelle . it is therefore attractive to propose that the premature death of dopaminergic neurons in the snc might be precipitated by a failure to control calcium homeostasis linked to a compromise of mitochondrial function . as mentioned above , neurons are specialised cells that have foregone the capacity to undergo cell division in the adult brain . as previously stated , however , mitochondria can also bring about cellular demise through apoptosis . it is therefore essential to keep the destructive power of this organelle under strict control . it is thought to play a role in the progression of neurodegenerative disease in humans . thus , it is conceivable that mitochondrial dysfunction may , at an early stage , trigger deficits in neurons prior to synapse formation , resulting in loss of connectivity , whereas overwhelming damage might induce apoptosis and cause neuronal death ( fig . 1 ) . neurons are large , specialised cells that rely on mitochondria for the majority of their energetic needs . a healthy mitochondrial population ensures the delivery of atp as a source of energy and acts as a calcium buffering system , decreasing the concentration of this ion in the cytosol . at synapses , defective mitochondria are likely to be less capable of producing atp and buffering calcium , possibly affecting efficient synaptic transmission . if these mitochondrial defects go unchecked , mitochondria will ultimately release pro - apoptotic factors , and , if this process occurs in the vicinity of the cell body , it is likely to result in neuronal death through apoptosis consequences of mitochondrial dysfunction in neurons . neurons are large , specialised cells that rely on mitochondria for the majority of their energetic needs . a healthy mitochondrial population ensures the delivery of atp as a source of energy and acts as a calcium buffering system , decreasing the concentration of this ion in the cytosol . at synapses , defective mitochondria are likely to be less capable of producing atp and buffering calcium , possibly affecting efficient synaptic transmission . if these mitochondrial defects go unchecked , mitochondria will ultimately release pro - apoptotic factors , and , if this process occurs in the vicinity of the cell body , it is likely to result in neuronal death through apoptosis parkinson s disease ( pd ) is a common neurodegenerative disease characterised initially by the loss of dopaminergic neurons in the substantia nigra and later by a more widespread loss of other neurons in the brain . between 10% and 15% of pd patients , however , have a family history of the disease , indicating that there is a strong genetic basis for this disease in this subgroup . the molecular pathogenesis of sporadic pd and the basis for selective dopaminergic neuronal loss remains unknown , and it is unclear whether gene mutations are involved in the development of this disease in this subgroup of patients . epidemiological studies consistently link exposure to pesticides to a higher incidence of pd . in particular , pesticides such as rotenone and paraquat have been shown to cause pd - like conditions in rodent models . in addition , the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) was shown to be responsible for the onset of severe pd - like symptoms in a group of young drug users in the 1980s . it is clear that such drugs affect the etc in mitochondria , resulting in loss of atp and perhaps an increase in free - radical generation . this evidence suggests that exposure to mitochondria - damaging agents might be important in the aetiology of pd in the sporadic patient population . even though mitochondrial dysfunction has been inconclusively linked to pd in the past few decades , genetic evidence indicating mitochondrial involvement in this disease a major breakthrough occurred in a recent study in which researchers identified disease - causing mutations in pten - induced putative kinase 1 ( pink1 ) in familial pd . more recently , mutations in the mitochondrial serine protease htra2 were reported to be associated with pd . pink1 is a putative mitochondrial kinase that acts to protect neurons from oxidative stress . in addition , it controls mitochondrial morphology in mammals and insects ( reviewed in thomas et al . ) . although animal models of pd fail to recapitulate all of the features of pd pathology in humans , they have proven to be invaluable tools for elucidating some of the mechanisms through which pink1 affects mitochondrial function . although the currently accepted models suggest that pink1 is a kinase , its direct substrates have so far remained elusive . perhaps the best evidence to indicate that the kinase activity of pink1 is required for its protective function comes from studies from drosophila melanogaster . in these studies , it was shown that a kinase - inactive version of pink1 failed to rescue the phenotypical features of pink1 loss - of - function flies . the fruit fly has proven to be an important tool in the dissection of the molecular determinants of pink1 function . these studies , however , have presented conflicting data regarding the importance of this gene for the survival of dopaminergic neurons ( see [ 16 , 17 ] ) . recently , it was reported that a loss of pink1 in mice leads to a significant decrease in dopamine release and a subsequent impairment of striatal synaptic plasticity , even in the absence of dopaminergic neuronal degeneration . in drosophila neurons loss of pink1 affects mitochondrial function and leads to a decrease in the atp levels that prevents synaptic vesicles from being mobilised during rapid stimulation . in addition , loss of pink1 in mice may also affect synaptic activity due to defects in mitochondrial calcium handling . abramov and colleagues proposed that pink1 regulates calcium efflux from mitochondria via the mitochondrial na / ca exchanger . deregulation of mitochondrial calcium handling can result in mitochondrial calcium overload that affects synaptic function . such an overload leads to increased levels of reactive oxygen species ( ros ) , which can then lead to mitochondria - dependent apoptosis . altogether , despite the limitations of pd models in non - primate animal species , both mice and flies have provided us with valuable clues regarding the mechanisms through which pink1 regulates mitochondrial function . neurons require mitochondria to function , and in the past few years , numerous findings have suggested that disruptions of mitochondrial function and dynamics contribute to ageing and neurodegenerative diseases ( recently reviewed in de castro et al . ) . the emerging view is that eukaryotic cells have developed exquisite mechanisms to cope with the diverse challenges imposed on mitochondrial integrity ( fig . 2 ) . mitochondria are therefore thought to have at least two levels of defence mechanisms that ensure their integrity and viability in individual cells ( reviewed in tatsuta and langer ) . the first line of defence is comprised of highly specific molecular quality control machinery composed of molecular chaperones and energy - dependent proteases that monitor the folding and assembly of mitochondrial proteins . this mitochondrial stress signalling results in the selective transcriptional up - regulation of nuclear genes encoding for mitochondrial hsp70 and hsp60 in cells experiencing elevated mitochondrial protein - conformational stress , therefore suggesting the existence of an unfolded protein response of mitochondrial origin ( upr ) . one of the major upstream components of this signalling pathway in the worm caenorhabditis elegans has been recently found to be the orthologue of escherichia coli clpp , a molecule that functions in the bacterial heat - shock response ( reviewed in broadley and hartl ) . these interesting observations suggest that mitochondria , as descendants of the original endosymbiont , have co - opted certain molecules of bacterial origin to stress signalling functions . mitochondria are dynamic organelles that are continuously remodelled by fusion and fission events . in this figure , mitochondrial fission is represented as one step of the mitochondrial life cycle involved in the maintenance of a pool of viable mitochondria . in this model , mitochondria can also employ fission to segregate defective daughter mitochondria based on a mechanism that requires opa1 . they can be repaired through molecular quality control pathways that possibly involve p38 , pink1 and htra2 . the defective mitochondria can also be selectively removed through autophagy using a mechanism that involves the pink1-dependent recruitment of the ubiquitin ligase parkin ( 1 ) , followed by the ubiquitination of proteins such as vdac1 and mfn2 ( orange and green colours , respectively ; purple ubiquitin ) ( 2 ) . such ubiquitinated proteins might then work as adaptors for p62/sqstm1 and promote the translocation of defective mitochondria to the autophagosome . finally , if both molecular and organellar quality control mechanisms fail , mitochondria will ultimately release pro - apoptotic proteins ( as indicated on the upper right of the figure ) , leading to cell death dynamics of mitochondrial quality control . mitochondria are dynamic organelles that are continuously remodelled by fusion and fission events . in this figure , mitochondrial fission is represented as one step of the mitochondrial life cycle involved in the maintenance of a pool of viable mitochondria . in this model , mitochondria can also employ fission to segregate defective daughter mitochondria based on a mechanism that requires opa1 . . they can be repaired through molecular quality control pathways that possibly involve p38 , pink1 and htra2 . the defective mitochondria can also be selectively removed through autophagy using a mechanism that involves the pink1-dependent recruitment of the ubiquitin ligase parkin ( 1 ) , followed by the ubiquitination of proteins such as vdac1 and mfn2 ( orange and green colours , respectively ; purple ubiquitin ) ( 2 ) . such ubiquitinated proteins might then work as adaptors for p62/sqstm1 and promote the translocation of defective mitochondria to the autophagosome . finally , if both molecular and organellar quality control mechanisms fail , mitochondria will ultimately release pro - apoptotic proteins ( as indicated on the upper right of the figure ) , leading to cell death once molecular quality control is overwhelmed within mitochondria , a second line of defence , termed organellar quality control , is thought to take over . this mechanism relies on the dynamic nature of mitochondrial populations to ensure the disposal of defective components . mitochondrial dynamics are thought to be important in the control of mitochondrial turnover and bioenergetic efficiency . the combined functions of fusion , fission and autophagy are now emerging as essential organellar quality control mechanisms that promote the sequestration , sorting and elimination of functionally impaired mitochondria . finally , if both of these lines of defence fail , severe mitochondrial damage unleashes the apoptosis pathway that culminates with cell death . is it possible that the demise of dopaminergic neurons in pd results from a failure of either one of the two described quality control mechanisms in mitochondria ? a positive answer to this question is starting to emerge from recent data derived from genetic studies in model organisms . xthe first link between pd and mitochondrial molecular quality control was suggested by the finding of heterozygous missense mutations in htra2 in sporadic cases of pd . the serine protease htra2 was initially identified as a mammalian homologue of the e. coli proteases htra / degp and degs . the structural and biochemical similarity of mammalian htra2 to the bacterial degs protease is particularly striking [ 2628 ] . in gram - negative bacteria , the degs protease is involved in a signal transduction pathway that senses and responds to envelope stress ( reviewed in ruiz and silhavy ) . in mammalian cells , given the similarities between degs and htra2 and taking into account that bacteria are the most likely ancestors of mitochondria , it is possible that htra2 evolved from degs and now controls the levels of unfolded proteins in mitochondria . the characterisation of mice lacking htra2 showed that loss of this protease results in a neurodegenerative phenotype with parkinsonian features . this phenomenon , however , is not due to the loss of dopaminergic neurons ; it is instead caused by the loss of a subpopulation of striatal neurons . interestingly , mitochondria lacking htra2 show increased levels of unfolded components of the etc , indicating that this protease is an important component of the molecular quality control in mitochondria and that disturbances in related quality control mechanisms might underlay parkinsonian neurodegeneration . in bacteria , studies focusing on the mechanisms of activation of mammalian htra2 have shown that this protease is likely to be activated through pink1-dependent phosphorylation . these observations , however , have failed to demonstrate direct phosphorylation of htra2 by pink1 and suggest that pink1 might instead act as a mitochondrial adaptor for the p38 stress - activated kinase . plun - favreau et al . showed that pink1 is involved in the phosphorylation of htra2 ; however , this protease is unlikely to be a direct target of the putative kinase activity of pink1 . taken together , these findings suggest that htra2 is possibly a downstream effector of pink1 in a mitochondrial molecular quality control pathway . the emerging picture suggests that pink1 is a modulator of not only molecular but also organellar quality control in mitochondria . surprisingly , it was shown that pink1 is capable of recruiting parkin , a cytosolic ubiquitin ligase , to damaged mitochondria [ 32 , 33 ] and targeting these organelles for autophagic clearance . the importance of parkin as a downstream effector of pink1 has been further confirmed from a series of impressive genetic studies in drosophila that showed that expression of parkin could rescue the phenotype of pink1 mutant flies [ 16 , 17 , 34 ] . ubiquitin has been suggested to act as a signal for the selective autophagy of diverse cargos , including protein aggregates , ribosomes , peroxisomes and pathogens in mammalian cells . it seems that , in damaged mitochondria , pink1 recruits parkin to catalyse the ubiquitination of proteins close to the outer mitochondrial membrane . two of the recently identified mitochondrial substrates for parkin are the voltage - dependent anion channel 1 ( vdac1 ) and mitofusin ( mfn ) . it remains to be determined if these are specific substrates for parkin or , upon pink1-dependent mitochondrial recruitment , if parkin simply labels any outer membrane protein with ubiquitin . the mammalian adaptor protein p62/sqstm1 ( sequestosome-1 ) is an autophagy receptor that can interact with ubiquitin conjugated to a target protein as well as lc3/gabarap on autophagosomes . it is therefore possible that ubiquitinated proteins on the outer mitochondrial membrane use p62/sqstm1 as an adaptor to anchor defective mitochondria to the autophagosome . this model is supported by a recent study in which p62/sqstm1 was found to be recruited to clustered mitochondria and to be essential for their final clearance . taken together , the findings to date suggest that pink1 is a master regulator of mitochondrial quality control , at least at two levels ; it enhances molecular quality control via htra2 and enhances organellar quality control via parkin . it is worth noting that mutations of all of these molecular components of mitochondrial quality control have been linked to pd , suggesting that alternative hits on this common pathway are possibly important in the development and progression of this disease . it is clear that unfolded proteins play an important role in the development of pd . although clear mechanisms for such proteinopathies are well established when these unfolded proteins accumulate in the cytosol , cell nucleus , endoplasmic reticulum and extracellular space , little is known about any causative role of protein aggregation in mitochondria in pd . although they have limitations , animal models are beginning to unravel a significant role for this phenomenon in pd when pink1-mediated mitochondrial quality control is impaired . the molecular pathogenesis of sporadic pd and the basis for selective dopaminergic neuronal loss remains unknown , and it is unclear if there is a genetic basis for the development of this disease in this subgroup of patients . recently , several genome - wide association studies ( gwas ) have been conducted to evaluate the possible genetic basis for pd in sporadic patients . to date , such approaches have failed to identify any genes associated with mitochondrial dysfunction in evaluated patients . given that multiple low - frequency risk variants are below the detection limit of current gwas methodology ( see ) , it is unclear whether mitochondrial gene mutations play any important role in the aetiology of pd in sporadic patients . with the increasing power of gene sequencing technologies , however , it is likely that , if other mitochondrial mutations in pd patients exist , they will come to light in the near future . approximately 40 years ago , us president richard nixon declared war on cancer by increasing research funding to improve the understanding of cancer biology . at first glance , neurodegeneration can be viewed as resulting from the death of postmitotic neurons , whereas cancer cells show an enhanced resistance to cell death . it is now clear that mitochondria are the master controllers of cell survival and death . interestingly , mitochondrial pink1 has been recently shown to be important as a survival mechanism downstream of the pi3k / akt signalling pathway . this pathway is often overactive in cancer cells , suggesting that pink1 could have oncogenic potential through the protection of healthy mitochondria in cancer cells . therefore , we reason that understanding the function of pink1 might help to fight the war on two fronts , as this knowledge will contribute to the elucidation of the role of mitochondria in both cancer and neurodegeneration . by increasing our knowledge of the power of mitochondria to control cellular life and death , modern scientists can be compared to the jedi knights of the fictional star wars galaxy , who harnessed the power of midichlorians to fight the dark side. we hope that , eventually , the dark side will not prevail and that such knowledge will one day translate into cures for devastating diseases such as pd .
recent findings from genetic studies suggest that defective mitochondrial quality control may play an important role in the development of parkinson 's disease ( pd ) . such defects may result in the impairment of neuronal mitochondria , which leads to both synaptic dysfunction and cell death and results in neurodegeneration . here , we review state - of - the - art knowledge of how pathways affecting mitochondrial quality control might contribute to pd , with a particular emphasis on the molecular mechanisms employed by pten - induced putative kinase 1 ( pink1 ) , htra2 and parkin to regulate mitochondrial quality control .
You are an expert at summarizing long articles. Proceed to summarize the following text: selective immunoglobulin a ( iga ) deficiency is the most common inherited immunodeficiency disorder worldwide . it is diagnosed when the total iga concentration is below 0.050.07 g / l in children over four years of age and when igg and igm are normal [ 13 ] . the global prevalence of iga deficiency varies from 1:134 to 1:18,500 depending on the population in question ; however , in many countries , as in lithuania , the prevalence is still unknown . approximately 90% of individuals with iga deficiency have no specific symptoms or signs . notwithstanding this often subclinical presentation , they carry a markedly increased risk of infections and long - term complications [ 79 ] . the risk of autoimmune diseases is also increased , in particular celiac disease , which is ten times more common in individuals with iga deficiency than in the general population . celiac disease often goes unrecognized in these patients because they do not express iga class transglutaminase 2 antibodies ( tg2-ab ) and are thus missed in routine screening . in fact , approximately 10% of iga - deficient blood donors may have unrecognized celiac disease . iga - deficient individuals may also develop anti - iga antibodies , which cause anaphylactic reactions upon transfusion of iga - containing blood products . large - scale laboratory - based testing is , however , impractical for this purpose . interestingly , a rapid point - of - care test for celiac disease requiring only a fingertip sample has recently been developed . this sensitive test simultaneously measures the presence of blood iga and could thus be used for the screening of iga deficiency in outpatient settings . we sought to establish the utility of the rapid fingertip test in population - based screening for selective iga deficiency among lithuanian schoolchildren . at the same time we were able to assess the previously unknown prevalence and clinical presentation of the condition in this population . the study was carried out in vilnius university children s hospital and the vilnius university faculty of medicine , lithuania , from january 2009 to march 2010 . six separate secondary schools in the hospital catchment area were randomly selected for study enrolment . altogether , 1583 fifth- to seventh - grade students ( 746 girls and 837 boys ) aged 1113 years were asked to participate in voluntary testing for iga deficiency and celiac disease by a well - instructed fifth - course medical student from the vilnius university medical faculty . of these subjects , a total of 1038 ( 65.5% ) parents and 1026 ( 64.8% ) children gave written informed consent to participate . only children with both personal and parents agreement twenty - six of the consenting pupils were absent from school at the time of the investigation . thus , exactly 1000 children ( 496 girls and 504 boys ) comprised the final cohort . the study protocol and recruitment were approved by the lithuanian bioethics committee . all procedures involving human participants were in accordance with the ethical standards of the committee and with the 1964 helsinki declaration and its later amendments . the presence of iga deficiency was investigated by a commercial point - of - care test ( biocard celiac test , ani biotech , vantaa , finland ) according to the manufacturer s instructions . the biocard test is a combined qualitative immunochromatographic assay measuring simultaneously the presence of blood iga and celiac disease - specific iga class tg2-ab . cutoff for negative iga is set at a blood value below 0.2 g / l . during the investigation , 10 microliters of blood were taken from the child s finger with a capillary tube , placed in a test tube with buffer solution . and shaken in order to mix the capillary blood with the buffer . next , three drops of the mixture were taken from the test tube and dropped into the pit of a disposable chromatographic plate , and results were assessed after 25 minutes . if a child was iga sufficient , one control strip appeared in the chromatographic field ; if the test field remained clear , the child was suspected of iga deficiency . the biocard point - of - care test has previously been shown to have 78.1% sensitivity and 100% specificity for biopsy - proven celiac disease and 96.7% sensitivity and 93.5% specificity compared with the serum tg2-ab and endomysial antibody tests from stored patient samples . if the presence of iga deficiency was suspected , the parents were informed and the child was referred to vilnius university children s hospital for thorough clinical investigation and further laboratory sampling . the serum iga , igm , igg , and ige concentrations were measured in the hospital laboratory using an immunoturbidimetric assay with routine methodology ( analyzer : cobas integra 400 plus , roche , switzerland ; reagents : roche diagnostics gmbh , germany ) . in addition , the total b and t cell counts were examined as part of hospital routine to exclude other immune deficiencies . according to the recommendations of north american and european guidelines , in addition , their health records were acquired from primary health care with permission , and the case histories were further compared with 30 age- and sex - matched iga - competent controls randomly selected from the original study cohort . besides routine clinical data , the frequency and presence of complicated disease courses ( in particular , gastrointestinal tract infections , upper and lower respiratory tract infections , diseases , allergic reactions , and autoimmune diseases ) were registered , as well as possible adverse reactions to vaccines and blood products . at time of the study 112,990 children aged 1113 years lived in lithuania ; of these , 15,138 lived in vilnius . because we aimed to establish the prevalence of iga deficiency with 90% calculated probability , it was necessary to study 1061 children in the republic of lithuania and 1001 in vilnius . with an estimated participation rate of 60% the study data were analyzed using epi info statistics ( centers for disease control and prevention , atlanta , georgia , usa ) and statistical package for the social sciences software ( spss inc . , the prevalence rate was calculated by writing the number of cases in the numerator and the figure for the whole population in the denominator . the study was carried out in vilnius university children s hospital and the vilnius university faculty of medicine , lithuania , from january 2009 to march 2010 . six separate secondary schools in the hospital catchment area were randomly selected for study enrolment . altogether , 1583 fifth- to seventh - grade students ( 746 girls and 837 boys ) aged 1113 years were asked to participate in voluntary testing for iga deficiency and celiac disease by a well - instructed fifth - course medical student from the vilnius university medical faculty . of these subjects , a total of 1038 ( 65.5% ) parents and 1026 ( 64.8% ) children gave written informed consent to participate . only children with both personal and parents agreement twenty - six of the consenting pupils were absent from school at the time of the investigation . thus , exactly 1000 children ( 496 girls and 504 boys ) comprised the final cohort . the study protocol and recruitment were approved by the lithuanian bioethics committee . all procedures involving human participants were in accordance with the ethical standards of the committee and with the 1964 helsinki declaration and its later amendments . the presence of iga deficiency was investigated by a commercial point - of - care test ( biocard celiac test , ani biotech , vantaa , finland ) according to the manufacturer s instructions . the biocard test is a combined qualitative immunochromatographic assay measuring simultaneously the presence of blood iga and celiac disease - specific iga class tg2-ab . cutoff for negative iga is set at a blood value below 0.2 g / l . during the investigation , 10 microliters of blood were taken from the child s finger with a capillary tube , placed in a test tube with buffer solution . and shaken in order to mix the capillary blood with the buffer . next , three drops of the mixture were taken from the test tube and dropped into the pit of a disposable chromatographic plate , and results were assessed after 25 minutes . if a child was iga sufficient , one control strip appeared in the chromatographic field ; if the test field remained clear , the child was suspected of iga deficiency . the biocard point - of - care test has previously been shown to have 78.1% sensitivity and 100% specificity for biopsy - proven celiac disease and 96.7% sensitivity and 93.5% specificity compared with the serum tg2-ab and endomysial antibody tests from stored patient samples . if the presence of iga deficiency was suspected , the parents were informed and the child was referred to vilnius university children s hospital for thorough clinical investigation and further laboratory sampling . the serum iga , igm , igg , and ige concentrations were measured in the hospital laboratory using an immunoturbidimetric assay with routine methodology ( analyzer : cobas integra 400 plus , roche , switzerland ; reagents : roche diagnostics gmbh , germany ) . in addition , the total b and t cell counts were examined as part of hospital routine to exclude other immune deficiencies . according to the recommendations of north american and european guidelines , selective iga deficiency was diagnosed if the iga concentration was less than 0.07 g / l and other immunoglobulins and b / t cell counts were normal . in addition , their health records were acquired from primary health care with permission , and the case histories were further compared with 30 age- and sex - matched iga - competent controls randomly selected from the original study cohort . besides routine clinical data , the frequency and presence of complicated disease courses ( in particular , gastrointestinal tract infections , upper and lower respiratory tract infections , diseases , allergic reactions , and autoimmune diseases ) were registered , as well as possible adverse reactions to vaccines and blood products . at time of the study 112,990 children aged 1113 years lived in lithuania ; of these , 15,138 lived in vilnius . because we aimed to establish the prevalence of iga deficiency with 90% calculated probability , it was necessary to study 1061 children in the republic of lithuania and 1001 in vilnius . with an estimated participation rate of 60% , six schools with the study data were analyzed using epi info statistics ( centers for disease control and prevention , atlanta , georgia , usa ) and statistical package for the social sciences software ( spss inc . , the prevalence rate was calculated by writing the number of cases in the numerator and the figure for the whole population in the denominator . the testing procedure was considered easy by both tester and children , and no adverse effects apart from minor fingertip pain were reported . the distribution of the study cohort and the presence of iga deficiency according to age and sex are shown in table 1 . there were no significant differences between the three age cohorts in the number of children or gender distribution . based on the biocard test , iga deficiency was suspected in one girl and one boy aged 12 years and in two boys aged 13 years . in subsequent laboratory analysis further , serum igm , igg , and ige concentrations as well as b / t cell counts were normal , and the children were not on any medication nor suffered from any other medical condition explaining the finding . consequently , all four received a diagnosis of selective iga deficiency ( table 1 ) . analysis of the health records of subjects with newly identified iga deficiency showed that all four had suffered from common infections such as otitis , sinusitis , tonsillitis , nasopharyngitis , laryngitis , or bronchitis from two to four times a year ( mean frequency 3.0 per year ) ; the frequency of infections did not differ from that in the iga - competent controls ( mean frequency of corresponding respiratory and ear infections 3.1 per year ; data not shown ) . none of the four iga - deficient children had suffered from documented gastrointestinal infection , but all had a history of chickenpox and one also of rubella . there were no reported complications after the infections , and antibiotics were used in a single case only for tonsillitis . none of the cases with iga deficiency had major allergies or adverse reactions to vaccines or blood products . the girl with iga deficiency had a previous diagnosis of psoriasis ( her parents did not suffer from this ) . the iga - deficient children reported no clinical symptoms or signs suggestive of celiac disease or other gastrointestinal disease , but upper gastrointestinal endoscopy with small - bowel mucosal biopsies was offered to all four according to our clinical practice at the time of the study . the endoscopy was performed on two children , which in both cases yielded normal macroscopic and microscopic findings ; the other two iga - deficient children declined the procedure . two of the 1000 study children ( 0.2% ) had normal iga and positive tg2-ab testing results . one ( a girl ) already had a previous celiac disease diagnosis but was not following a strict gluten - free diet . the other ( a boy ) was biopsied and found to have celiac disease ; he was subsequently placed on a gluten - free diet . the main finding in the present study was that the rapid point - of - care fingertip test is an easy , practical , and accurate method to screen for selective iga deficiency in an unselected pediatric population . furthermore , the previously unknown prevalence of iga deficiency among lithuanian children was now found to be 1:250 . the main function of iga is mucosal defense in the airways and intestine , where the body encounters most pathogens and thus needs to mount a fast and effective immunological response . accordingly , iga - deficient patients run an increased risk , in particular , of different respiratory and gastrointestinal tract infections [ 710,12,17,18 ] . healing of the infections may also be compromised , and complications such as chronic diarrhea and bronchiectasis may follow . here we found no difference in the frequency or severity of infections between iga - deficient children and controls . this in itself is no surprise , because these problems usually manifest later in adulthood . however , there is wide individual variation , and some patients may develop severe infections in infancy . this heterogeneity in clinical presentation might be explained by replacement of iga functions by other immunoglobulins in some individuals , while in others there might be concomitant deficiencies , for example , in certain igg subclasses . in any case , an early diagnosis of the condition enables prompt recognition of severe infections and hence lowers the risk of long - term complications . none of the iga - deficient children in this study was found to have celiac disease , but only two were biopsied and we were not able to measure igg - class antibodies . the marked overrepresentation of celiac disease in patients with iga deficiency is clinically important , because recent studies have shown it to be one of the commonest pediatric disorders . because iga - based tests are used in first - line screening for celiac disease , iga - deficient subjects are easily missed . these unrecognized cases have , besides the burden of symptoms and unnecessary investigations , an increasing risk of complications such as anemia and poor growth . in these circumstances the rapid test is of great value in allowing simultaneous investigation of the presence of both celiac disease and iga deficiency . besides celiac disease , iga - deficient individuals are also at risk of other autoimmune diseases such as rheumatoid arthritis and myasthenia gravis . in the present study the only girl with iga deficiency had psoriasis . this might be a coincidence , but such a combination has previously been reported , and it is possible that iga deficiency predisposes to the development of this disorder . another issue advocating early diagnosis of iga deficiency is the risk of anaphylactic reactions to transfusion products such as red blood cells and platelets . it has been suggested that such reactions can be prevented by providing blood components lacking iga collected from iga - deficient donors . based on this option , donor registries have been established in the usa and canada , and also in some countries in europe and asia [ 2830 ] . the rapid test could be particularly useful in this setting , on the one hand in excluding iga deficiency in subjects receiving blood products and on the other hand in screening for possible donors for the registries . for the first time , we were able to establish the prevalence of iga deficiency in the lithuanian population , showing it to be 0.4% . this result is both nationally and ethnically representative , because the country s population is more than 80% ethnic lithuanian , and 95% of our cohort were lithuanians . a comparison with worldwide prevalence figures is presented in table 2 . in general , the lithuanian prevalence is fairly high , but then again it is close to that in neighboring countries such as estonia , sweden , and finland . however , it must be emphasized that in many previous studies only blood donors or hospital - based cohorts were examined , and this may not accurately reflect the true prevalence ( table 2 [ 3243 ] ) . in contrast , using the rapid test we were able to screen an unbiased sample of the pediatric population . the major strength of the present study is the well - defined and nationally representative study cohort . an obvious limitation is the small number of iga - deficient cases , which reduced statistical accuracy and rendered the comparisons of medical histories less useful . in theory it is also possible that we missed some iga - deficient cases , because not all study children underwent laboratory testing . that being said , in this study the tests were read by an experienced reader ; the evaluation might be less reliable if undertaken by a layman . finally , our results should not be generalized to very young children , who may have physiologically low iga values . the rapid point - of - care fingertip test significantly reduces the time and costs associated with population screening for iga deficiency , and is also an accurate and practical means of case finding in outpatient settings . the prevalence of iga deficiency in the lithuanian pediatric population is close to that seen in neighboring baltic and scandinavian countries .
backgroundselective immunoglobulin a ( iga ) deficiency is the most common inherited immunodeficiency disorder world - wide . an early diagnosis is advocated because of the increased risk of infections , autoimmune diseases , and allergic reactions . we investigated the usefulness of a rapid point - of - care test in detecting for iga deficiency in a population with a previously unknown prevalence.material/methodsaltogether , 1000 children aged 1113 years from randomly selected lithuanian schools were enrolled . a point - of - care test with a fingertip sample was used to screen for the presence of iga deficiency in children whose parents gave consent . those with suspected iga deficiency were referred to hospital for further clinical examination and confirmation of the diagnosis . in addition , their medical histories were compared with those of 30 age- and sex - matched healthy controls.resultsiga deficiency was suspected in one girl and in three boys on the basis of the rapid test , and the diagnosis was confirmed for all four cases ( prevalence 0.4% , 95% confidence interval 0.161.02% ) . there was no difference in disease history or complications between iga - deficient children and healthy controls.conclusionsthe rapid antibody test is a practical and accurate method to diagnose selective iga deficiency in children . the prevalence of iga deficiency among lithuanian schoolchildren is 1:250 .
You are an expert at summarizing long articles. Proceed to summarize the following text: fibromuscular dysplasia ( fmd ) is a rare disease affecting small and middle - sized arteries of the kidney and brain , predominantly in adult women . it rarely occurs in children and has not previously been treated with thrombolytics following acute stroke . a 12-year - old girl was admitted to the pediatric emergency room after having collapsed during a soccer game . family members reported that she had fallen down with convulsions of all four extremities . on admission , a neurological examination revealed a severe left - hemispheric syndrome with global aphasia , conjugated gaze deviation to the left , severe hemiparesis with extensor plantar response and hemihypesthesia of the right side [ national institute of health stroke score ( nihss ) : 19 ] . both the family history and her medical history were unremarkable apart from a migraine without aura . acute cerebral mri showed an acute middle cerebral artery ( mca ) stroke on diffusion - weighted images involving the insula , the lentiform nucleus , the head of the nucleus caudatus and the centrum semiovale ( fig . 1a ) , with occlusion of distal m3 branches of the left mca and irregular contrasting of both the proximal left m1 and the distal left internal carotid artery ( ica ) segments ( fig . 1b ) revealed an extended perfusion deficit beyond the territory of the diffusion - weighted image lesions in the cortical areas of the complete mca territory , characteristic of a significant mismatch between the hypoperfused area and the diffusion impairment . the patient was treated within an extended time window of 5 h after onset of the symptoms with intravenous t - pa ( 0.9 mg / kg body weight , resulting in 5 mg as a bolus and 50 mg continuously over 60 min ) . forty - eight hours later , marginal improvement was observed ( nihss after thrombolysis : 15 ) . laboratory studies were unremarkable as were an extended coagulation profile , vasculitis screening and analysis of the cerebrospinal fluid . transthoracic and transesophageal echocardiography showed no abnormalities , while both extracranial and transcranial duplex sonography demonstrated a severe stenosis in the siphon of the left ica with significantly decreased prestenotic flow velocity . furthermore , the sonography results showed a retrograde flow direction in the left proximal anterior cerebral artery , indicating hemodynamic compensation from right to left anterior circulation . the size of the ischemia on a follow - up mri the next day was unchanged and without secondary hemorrhagic transformation . on mr angiography , all 3 mca segments , including the distal m3 branches , were recanalized while mild pathology within the proximal m1 and the distal ica persisted . digital subtraction angiography of these vessel segments showed typical constrictions with ' strings of beads ' characteristic of fmd ( fig . another follow - up mri at day 16 showed a new though clinically asymptomatic ischemic lesion in the left mca territory , which prompted oral anticoagulant treatment with phenprocoumon ( inr 2.0 - 3.0 ) . the patient was released into rehabilitation after further improvements ( nihss on discharge : 8) and presented with severe paresis of the right arm and moderate paresis of the right leg . three months later we saw her again with further clinical improvement ( nihss : 5 ) : only moderate right spastic hemiparesis and mild residual aphasia persisted . stroke in children is rare , with an incidence varying from 1.3 to 13.0 per 100,000 . while systemic thrombolysis with t - pa is considered the gold standard of acute treatment in adult ischemic stroke patients [ 2 , 3 ] , insufficient data on systemic intravenous or local intra - arterial thrombolysis in children are available [ 4 , 5 , 6 ] . a major problem is the delay in diagnosis of stroke , which is even more challenging than in adults , as this diagnosis is rarely assumed by parents , family members or doctors . although stroke in young adults is often due to similar causes as those in the elderly , active mechanisms in children are different . fmd is very rare in children and has only been reported in a few casual communications [ 8 , 9 , 10 ] . as mr angiography is unable to provide sufficient resolution to demonstrate small multisegmental structural changes of commonly multiple brain - supplying arteries , digital subtraction angiography is needed . therefore , fmd of the carotid or intracranial arteries often remains undiagnosed . the use of platelet inhibitors or oral anticoagulation for 6 - 12 months after clinical events in adults is a possible regimen in children as well . as a differential diagnosis , transient cerebral angiopathy ( tca ) was discussed . despite being frequently considered as a reason for stroke in children with focal stenosis of the intracranial vessels , the diagnosis of tca does not describe a particular underlying pathophysiology and , other than an association with varicella zoster infections , there is little known about its etiology . an overlap of tca with fmd can not be excluded . to our knowledge , this is the first report of a child with fmd treated with t - pa , and also the first one that describes thrombolysis of an acute stroke which was based on mri diffusion / perfusion mismatch criteria , in a child .
fibromuscular dysplasia , predominantly found in adult women , is a rare disease of small and middle - sized arteries of the kidney and brain . we present a case of a 12-year - old girl with acute ischemic stroke , due to fibromuscular dysplasia of the distal internal carotid artery and the proximal middle cerebral artery , which was successfully treated with t - pa .
You are an expert at summarizing long articles. Proceed to summarize the following text: osseointegrated dental implants have proven to be predictably successful when appropriate guidelines are followed . traditionally , implant treatment of edentulous patients was based on a two - stage surgical protocol , with a healing period of three to six months , during which the implants were submerged to achieve osseointegration . this approach was considered to be an essential step for successful implant treatment , as it was believed that the micromovement of the implants , due to functional forces at the bone - implant interface during wound healing , could induce the formation of fibrous tissue rather than bone , leading to failure . several aspects of the implant morphology have been studied for years . among these , a submerged implant was thought necessary to prevent infection and epithelial downgrowth . more recently , several reports have demonstrated that one - stage surgery , with immediate loading , is possible , with good clinical results . although zdis have been used since the nineties , there are few reports focusing on the clinical outcome of these fixtures . consequently , we decided to perform a retrospective study on a large series of zdis , to identify the variables statistically associated with the clinical outcome . in the period between january 2007 and june 2011 , 125 patients were treatetd with zdis by two surgeons ( cms and ez ) . the last check - up was performed in june 2012 , with a mean follow - up period of 17 9 months ( minimummaximum , 8 - 64 months ) . controlled oral hygiene , the absence of any lesions in the oral cavity , and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length . in addition , the patients had to agree to participate in a post`-operative check - up program . insufficient bone volume , a high degree of bruxism , smoking more than 20 cigarettes / day and excessive consumption of alcohol , localized radiation therapy of the oral cavity , antitumor chemotherapy , liver , blood , and kidney diseases , immunosupressed patients , patients taking corticosteroids , pregnant women , patients with inflammatory and autoimmune diseases of the oral cavity , and poor oral hygiene . before surgery , radiographic examinations were done with the use of an orthopantomograph and computed tomography ( ct ) scans . in each patient , the peri - implant crestal bone levels were evaluated by a calibrated examination of the ortopantomograph x - rays . measurements were recorded before surgery ; after surgery , and at the end of the follow - up period . the measurements were carried out mesially and distally to each implant , calculating the distance between the edge of the implant and the most coronal point of contact between the bone and the implant . the bone level recorded just after the surgical insertion of the implant was the reference point for the following measurements . a peak scale loupe with a magnifying factor of seven times and a scale graduated in 0.1 mm was used . peri - implant probing was not performed because controversy still existed with regard to the correlation between the probing depth and implant success rates . the implant success rate ( scr i.e. , good clinical , radiological , and esthetic outcomes ) was evaluated according to the following criteria : ( 1 ) absence of persisting pain or dysesthesia ; ( 2 ) absence of peri - implant infection with suppuration ; ( 3 ) absence of mobility ; and ( 4 ) absence of persisting peri - implant bone resorption greater than 1.5 mm during the first year of loading and 0.2 mm / year during the following years . in 125 patients a total of 556 plants were inserted : two hundred and ninety - five ( 53.1% ) in the maxilla and 261 ( 46.9% ) in the mandible . there were 480 ( 86.3% ) screw - vents [ figure 1 ] , 51 ( 9.2% ) swiss - plusses [ figure 2 ] , and 25 ( 4.5% ) splines [ figure 3 ] . sixteen , 355 , 34 , 90 , 55 , and 6 fixtures had a diameter of 3.25 , 3.7 , 3.75 , 4.1 , 4.7 , and 4.8 mm , respectively . twenty - eight , 145 , 5 , 217 , 8 , 141 , and 12 implants had a length of 8 , 10 , 11 , 11.5 , 12 , 13 , and 14 mm , respectively . the implants were inserted to replace 136 ( 24.5% ) incisors , 80 ( 14.4% ) cuspids , 198 ( 35.6% ) premolars , and 142 ( 25.5% ) molars . two surgeons inserted the implants : cms 324 ( 58.3% ) and ez 232 ( 41.7% ) . three hundred and twenty - seven ( 58.8.% ) were inserted in females and 229 ( 41.2 % ) in males . seven ( 1.3% ) implants were inserted in diabetic patients and 80 ( 14.4% ) in subjects treated with anti - hypertensive drugs . thirty - four ( 6.1 ) were inclinated as they were used for an all - on - four technique and 179 ( 32.2% ) were inserted in smokers ( less than 20 cigarettes per day ) . an antimicrobial prophylaxis was administered with 1 g amoxycillin twice daily for five days , starting one hour before surgery . local anesthesia was induced by infiltration of articaine / epinephrine and postsurgical analgesic treatment was performed with 100 mg nimesulid , twice daily , for three days . the sutures were removed seven days after surgery . in case of a two stage procedure , following 24 weeks of implant insertion , the provisional prosthesis was provided , and the final restoration was usually delivered within an additional eight weeks . the number of prosthetic units ( i.e. , implant / crown ratio ) was about 0.6 . the mean age was 62 10 years ( minimummaximum 23 - 88 years ) . two hundred and thirty - two ( 41.7% ) implants were insered after the use of piezo , laser , and platelet - rich plasma derivates . fourteen ( 2.5% ) were covered with resorbable membranes and 244 ( 43.9% ) were inserted in post - extractive sockets . as no implants were lost ( i.e. , srv = 100% ) and no statistical differences were detected among the studied variables , no or reduced crestal bone resorption was considered as an indicator of scr , to evaluate the effect of several host- , implant- , and occlusion - related factors . the difference between the implant abutment junction and the bone crestal level was defined as the implant abutment junction ( iaj ) and calculated at the time of operation and during follow - up . delta iaj was the difference between iaj at the last check - up and iaj recorded just after the operation . the pearson chi - sqaure test was used to detect the variables most associated with implant success . in the period between january 2007 and june 2011 , 125 patients were treatetd with zdis by two surgeons ( cms and ez ) . the last check - up was performed in june 2012 , with a mean follow - up period of 17 9 months ( minimummaximum , 8 - 64 months ) . controlled oral hygiene , the absence of any lesions in the oral cavity , and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length . in addition , the patients had to agree to participate in a post`-operative check - up program . insufficient bone volume , a high degree of bruxism , smoking more than 20 cigarettes / day and excessive consumption of alcohol , localized radiation therapy of the oral cavity , antitumor chemotherapy , liver , blood , and kidney diseases , immunosupressed patients , patients taking corticosteroids , pregnant women , patients with inflammatory and autoimmune diseases of the oral cavity , and poor oral hygiene . controlled oral hygiene , the absence of any lesions in the oral cavity , and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length . in addition , the patients had to agree to participate in a post`-operative check - up program . insufficient bone volume , a high degree of bruxism , smoking more than 20 cigarettes / day and excessive consumption of alcohol , localized radiation therapy of the oral cavity , antitumor chemotherapy , liver , blood , and kidney diseases , immunosupressed patients , patients taking corticosteroids , pregnant women , patients with inflammatory and autoimmune diseases of the oral cavity , and poor oral hygiene . before surgery , radiographic examinations were done with the use of an orthopantomograph and computed tomography ( ct ) scans . in each patient , the peri - implant crestal bone levels were evaluated by a calibrated examination of the ortopantomograph x - rays . measurements were recorded before surgery ; after surgery , and at the end of the follow - up period . the measurements were carried out mesially and distally to each implant , calculating the distance between the edge of the implant and the most coronal point of contact between the bone and the implant . the bone level recorded just after the surgical insertion of the implant was the reference point for the following measurements . a peak scale loupe with a magnifying factor of seven times and a scale graduated in 0.1 mm was used . peri - implant probing was not performed because controversy still existed with regard to the correlation between the probing depth and implant success rates . the implant success rate ( scr i.e. , good clinical , radiological , and esthetic outcomes ) was evaluated according to the following criteria : ( 1 ) absence of persisting pain or dysesthesia ; ( 2 ) absence of peri - implant infection with suppuration ; ( 3 ) absence of mobility ; and ( 4 ) absence of persisting peri - implant bone resorption greater than 1.5 mm during the first year of loading and 0.2 mm / year during the following years . in 125 patients a total of 556 plants were inserted : two hundred and ninety - five ( 53.1% ) in the maxilla and 261 ( 46.9% ) in the mandible . there were 480 ( 86.3% ) screw - vents [ figure 1 ] , 51 ( 9.2% ) swiss - plusses [ figure 2 ] , and 25 ( 4.5% ) splines [ figure 3 ] . sixteen , 355 , 34 , 90 , 55 , and 6 fixtures had a diameter of 3.25 , 3.7 , 3.75 , 4.1 , 4.7 , and 4.8 mm , respectively . twenty - eight , 145 , 5 , 217 , 8 , 141 , and 12 implants had a length of 8 , 10 , 11 , 11.5 , 12 , 13 , and 14 mm , respectively . the implants were inserted to replace 136 ( 24.5% ) incisors , 80 ( 14.4% ) cuspids , 198 ( 35.6% ) premolars , and 142 ( 25.5% ) molars . two surgeons inserted the implants : cms 324 ( 58.3% ) and ez 232 ( 41.7% ) . three hundred and twenty - seven ( 58.8.% ) were inserted in females and 229 ( 41.2 % ) in males . seven ( 1.3% ) implants were inserted in diabetic patients and 80 ( 14.4% ) in subjects treated with anti - hypertensive drugs . thirty - four ( 6.1 ) were inclinated as they were used for an all - on - four technique and 179 ( 32.2% ) were inserted in smokers ( less than 20 cigarettes per day ) . an antimicrobial prophylaxis was administered with 1 g amoxycillin twice daily for five days , starting one hour before surgery . local anesthesia was induced by infiltration of articaine / epinephrine and postsurgical analgesic treatment was performed with 100 mg nimesulid , twice daily , for three days . the sutures were removed seven days after surgery . in case of a two stage procedure , following 24 weeks of implant insertion , the provisional prosthesis was provided , and the final restoration was usually delivered within an additional eight weeks . the number of prosthetic units ( i.e. , implant / crown ratio ) was about 0.6 . the mean age was 62 10 years ( minimummaximum 23 - 88 years ) . two hundred and thirty - two ( 41.7% ) implants were insered after the use of piezo , laser , and platelet - rich plasma derivates . fourteen ( 2.5% ) were covered with resorbable membranes and 244 ( 43.9% ) were inserted in post - extractive sockets . as no implants were lost ( i.e. , srv = 100% ) and no statistical differences were detected among the studied variables , no or reduced crestal bone resorption was considered as an indicator of scr , to evaluate the effect of several host- , implant- , and occlusion - related factors . the difference between the implant abutment junction and the bone crestal level was defined as the implant abutment junction ( iaj ) and calculated at the time of operation and during follow - up . delta iaj was the difference between iaj at the last check - up and iaj recorded just after the operation . the pearson chi - sqaure test was used to detect the variables most associated with implant success . the mean peri - implant bone resorption was 0.7 0.6 mm ( minimum - maximum 0 - 5.7 mm ) . twenty - seven fixtures had a peri - implant bone resorption greater than the cut - off values ( scr = 95.1% ) , and therefore , were used to detect those variables statistically related to an augmented peri - implant bone resorption . among the studied variables ( i.e. , implant type , diameter , length , gender , smoke , diabetes , drugs , platelet - rich plasma , sinus augmentation , membrane , immediate loading , fixture inclination , laser , piezo , post - extraction socket , surgeon , tooth position , and jaws ) only jaws was statistically significant , with a better outcome for implants inserted in the maxilla ( p = 0.017 ) . zdis have been used since the nineties , but few reports analyzed the clinical outcome of these fixtures . in 2001 , khayat et al . reported a study on a wide diameter screw - vent . one hundred and eleven implants were evaluated at the recall examination . almost all implants ( 109 ) supported a fixed partial prosthesis . no implants were lost during the loading period . in 2002 , arlin analyzed 435 screw - vent implants . the focus group was compared to a mixed implant design group , with a variety of abutment connections and surfaces from several other manufacturers . the cumulative survival rates were 94.2% ( n = 435 ) for the focus group and 90.1% ( n = 2339 ) for the reference group . in 2005 , minichetti et al . reported the results of implants placed into extraction sites grafted with particulate mineralized bone allograft ( puros ) . a total of 313 extraction sites were grafted with mineralized bone graft during a 36-month period . a total of 252 screw - vent implants were placed into the grafted extraction sites after a four- to seven - month healing period . a total of 244 implants were restored with fixed prosthesis and six with removable overdentures , for a total of 250 loaded implants . a total of six implants failed , which required their removal ( two implants before load and four after loading ) , resulting in a 97.6% implant success rate . in 2007 , khayat et al . a total of 835 implants , with diameters of 3.7 mm ( 9% ) , 4.7 mm ( 76% ) , and 6.0 mm ( 15% ) were placed in 328 patients , using a single - stage , delayed - loading protocol . the implants were restored with a variety of prostheses and monitored over two years of functional loading . cumulative implant survival was 99.4% ( n = 835 ) ; differences between mandibular ( 99.0% , n = 408 ) and maxillary ( 99.8% , n = 427 ) implants were not statistically significant . six implants failed to meet the success criteria by sustaining mesial and distal bone loss below the first implant thread ; however , they remained stable and continued functioning without pain or inflammation . cumulative implant success was 98.6% ( n = 835 ) ; the differences between the maxillary ( 98.6% ) and mandibular ( 98.8% ) implants were not statistically significant . the success rates by implant diameter were 98.6% ( 3.7 mm ) , 98.4% ( 4.7 mm ) , and 100% ( 6 mm ) . the authors concluded that after two years of functional loading , the survival and success rates for screw - vent implants placed in a non - submerged protocol , equaled or surpassed those of single - thread , straight - walled implant historical controls . one year later , minichetti et al . reported the results of implants placed in the maxillary sinuses grafted with particulate mineralized cancellous bone allograft alone or in combination with resorbable hydroxyapatite , over a three - year period . a total of 56 sinuses were grafted , and 136 dental implants were placed into the grafted sites after a four- to eight - month healing period . all re - entries revealed a bony hard structure acceptable for osteotomy preparation . of these implants , 124 had been restored with fixed prosthesis and 12 with removable overdentures for a total of 136 loaded implants . a total of three implants required removal ( failure ) resulting in a 97.7% implant success rate ( 2.3% failure rate ) . the authors concluded that a mineralized human allograft placed in a lateral window sinus elevation , was a clinically predicable method , acceptable for implant placement and restoration . evaluated the immediate loading implant and possible effects correlated with peri - implants . in 2008 , ormianer et al . , performed a non - randomized , uncontrolled , retrospective study to evaluate the clinical outcomes of treatment with tapered , multithreaded implants , with a special emphasis on peri - implant crestal bone status . chart reviews were conducted of 60 patients who had been treated with 267 implants for the placement of one or more missing and/or unsalvageable teeth , and who met the general inclusion criteria for dental implant therapy . in all cases , marginal bone changes were calculated from the cementoenamel junction or the implant neck to the crestal bone level , with standardized radiographs taken at the implant placement ( baseline ) and during annual follow - up . after a mean follow - up of 7.5 years , the implant survival was 98.5% ( 263/267 ) for all implants placed , and the implant success was 96.2% ( 253/263 ) for all surviving implants . crestal bone loss was observed in 25% ( 15/60 ) of the total study subjects and in 12% ( 32/263 ) of all surviving implants : twenty - nine implants exhibited 1 mm of bone loss and three implants lost 2 mm of bone . low - density maxillary jawbone and more extensive bone remodeling , which were required around the implants immediately placed into extraction sockets , were the probable causes of the observed bone loss in this study . the implants exhibited excellent long - term outcomes , with little or no bone loss . most of the studied variables ( i.e. , implant type , diameter , length , gender , smoke , diabetes , drugs , platelet - rich plasma , sinus augmentetion , membrane , immediate loading , fixture inclination , laser , piezo , post - extraction socket , surgeon , and tooth position ) have no impact on the clinical outcome . implants inserted in the mandible have a slight , but a statistically significant worse outcome , with a higher peri - implant bone resorption , compared to those inserted in the maxilla . in conclusion , zdis are reliable devices to use in implantology .
background : one - stage surgery with immediate loading is possible , with good clinical results . many types of dental implants are available in the market . zimmer dental implants ( zdis ) have been used since the nineties , but few reports have analyzed the clinical outcome of these fixtures . we planned a retrospective study on a series 566 zdis , to evaluate their clinical outcome.materials and methods : in the period between january 2007 and june 2011 , 125 patients were treatetd with zdis . the last check - up was performed in june 2012 , with a mean follow - up period of 17 9 months ( minimum maximum , 8 - 4 months ) . zdis were inserted as follows : 295 ( 53.1% ) in the maxilla and 261 ( 46.9% ) in the mandible . there were 480 ( 86.3% ) screw - vents , 51 ( 9.2% ) swiss plusses , and 25 ( 4.5% ) splines . sixteen , 355 , 34 , 90 , 55 , and six fixtures had a diameter of 3.25 , 3.7 , 3.75 , 4.1 , 4.7 , and 4.8 mm , respectively . twenty - eight , 145 , 5 , 217 , 8 , 141 , and 12 implants hade a length of 8 , 10 , 11 , 11.5 , 12 , 13 , and 14 mm , respectively . the implants were inserted to replace 136 ( 24.5% ) incisors , 80 ( 14.4% ) cuspids , 198 ( 35.6% ) premolars , and 142 ( 25.5% ) molars.results:no implants were lost ( i.e. , srv = 100% ) . among the studied variables , only those for the jaws were statistically significant , with a better outcome for implants inserted in the maxilla ( p = 0.017).conclusions : zdis are reliable devices to be used in implantology , althougth a higher marginal bone loss has to be expected when these implants are inserted in mandible .
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Proceed to summarize the following text: many studies have been done on the design and stress distribution of removable partial dentures rpds and abutment teeth using finite element method ( fem ) , strain gauges , and photo elastic techniques [ 115 ] . appropriate design in a rpd is critical for proper force distribution and avoidance of excessive loading of the periodontal ligament ( pdl ) of the abutment tooth . by splinting an isolated abutment ( often termed a pier abutment or lone - standing tooth ) to its anterior tooth with an appropriately constructed fixed partial denture ( fpd ) , detrimental forces on abutment tooth will be minimized . however , these teeth may not be splinted with a fpd , due to the clinicians tendency of not preparing an intact isolated abutment and its anterior tooth . there is little scientific evidence about the consequences and differences of splinting or not splinting isolated teeth [ 24 ] . however , the clinician should determine ( on an individual basis ) whether the patient would benefit from splinting an isolated tooth with a fpd or not . in the dental community , there is no consensus in treatment planning for lone standing abutments . for example , a dentist choosing to place a direct retainer on a canine rather than a second premolar is considered to have made a poor choice from an esthetic viewpoint . however , if a dentist decides to clasp the second premolar ( i.e. isolated tooth ) , his or her choice may lead to generation of potentially detrimental forces . in addition , the preparation of an intact canine and second premolar for a fpd is neither conservative nor cost - effective for patients , but will reduce force to the pier abutment . in a kennedy class 2 design , use of a modification on the other side of the arch , even at the expense of keeping a pier abutment , improves prosthesis stability and retention . however , this abutment may be subject to torsional forces above its pdl s physiological tolerance . a mesial rest is one of the approved designs for reducing deleterious forces ( generally resulting from denture base movement ) . the mesial rest can change the fulcrum line location and help direct the forces more vertically to the residual ridge . in addition , a more flexible retentive clasp ( wrought wire ) can be used for minimizing harmful forces to the pier abutment . reciprocal parallel interface designed with a mesial rest , distal proximal plate , and i - bar clasp ( positioned at or mesial to the mesiodistal height of contour ) the amount of torsional forces applied to abutments depends on the prosthesis design , quality of the bone and soft tissue of the residual ridge , extension of free - end denture base , and patient s muscular forces . each abutment should be examined carefully for its bony support and previous bone reaction to occlusal stress . lack of consensus exists among different studies about the location of occlusal rest on the distal abutment tooth ( either mesially or distally ) . the purpose of this 3d fem study was to investigate the stress induced in the distal abutments of an i - bar - retained rpd in a mandibular bilateral distal extension partial denture where the distal pier abutments had varying locations of occlusal rests ( either mesially or distally ) and the other abutment was not isolated . using pre - existing ct scan data obtained from a 24 year - old man s skull , a 3d mandibular model was fabricated . in the model , there was 1 to 2 mm distance between ct section images . this software processes 3d images and converts ct data to 3d computer aided design ( cad ) models . in the modeling procedure , mimics software was used to eliminate the first premolar on one side and first and second molars of both sides , so that the second premolar was isolated between the two edentulous areas . in this way , a kennedy class 1 , modification 1 rpd was simulated in the mandible . by preparing dicom films , mimics software was used to isolate the trabecular bone , cortical bone and tooth crown with x - ray absorption coefficients of 100300 , 3002000 and more than 2000 , respectively . mimics software data were transferred to solid works 2009 ( structural research & analysis corporation , ca , usa ) for modeling and fem analyses . sockets of eliminated second premolars were filled with 18.5 mm thickness cancellous bone and covered with 1.5 mm thickness cortical bone . all the materials used in this study were considered to be linearly elastic , homogenous and isotropic . two different models were designed on this partially edentulous model with an isolated second premolar for comparison . in all models , a half - pear - shaped lingual bar with 5 mm height and 2 mm thickness , which allowed 3 mm of space between the gingival margins and the superior border of the bar was constructed as the major connector . the reciprocal parallel interface ( rpi ) design , with an i - bar retentive clasp positioned in the mid - buccal undercut and a mesial rest , was designed for the isolated second premolar . the contact area between i - bar and abutment tooth was about 2 mm in height and 1.5 to 2 mm in width . the second model was identical to the first model , with the exception of just positioning two mesial and distal rests on the isolated second premolar . in the third model , the first premolar was not eliminated and there was no isolated tooth . to compare stress concentration , other parts of the framework design were kept identical with the other two models such as the use of rpi design ( fig . simulation was performed with a static analysis by using cosmos works 2009 software with which , boundary conditions and physical properties of different components were defined more accurately [ 6,16 , 17 ] ( table 1 ) . the horizontal component of the i - bar was 4 mm farther from the gingival margin . in the distal of the isolated tooth , the proximal plate was extended to the cervical one - third of the abutment . in the opposite side of the arch , 1 ) . meshing of model 1 ( yellow represents mandibular bone , red and pink show the rpd framework and acrylic resin , respectively ) . since the analysis was static , it was independent from loading time and all contact border conditions between all parts were considered equal . in the pdl , contact elements with a node - to - node connection were used . the mesh size at the contact area ( i - bar - tooth ) should be small . sharp edges , rest seats and force application surfaces are other critical areas to be considered as stress concentration sites . the mesh size was 1.5 mm and a total of 393,314 elements and 637,348 nodes were used in this model ( figs . meshing of model 3 ( the i - bar and the enamel surface are connected with a surface - to - surface connection ) masseter and medial pterygoid muscles were considered as the main sites of application of masticatory forces . the occlusal force exerted against the complete denture , fpd , and rpd varies considerably . on the rpd , it is about 2526n [ 18 , 19 ] . in this study , 26 n load was exerted bilaterally on the first - molar sites , about 13 mm distal to the first premolar . the objective of this fem study was to compare the amount of stress and probable movement of an isolated mandibular second premolar tooth in two conventional rpi designs of rpds and the same rpd abutment tooth ( which was not isolated ) . in the first model ( isolated second premolar with a mesial rest ) , the von mises stress distribution at the contact area of i - bar clasp and cervical portion of tooth ( from buccal and distobuccal views ) was 19 mpa and the maximum stress generated in this area was 30 mpa ( with a range of 12 to 22 mpa ) . in this model , the mean strain was 90 microstrains and the maximum strain was 1550 microstrains . in the distal proximal plate , the von mises stress distribution was 13 mpa . in the mesial rest seat , the von mises stress distribution was 30 mpa ( with a range of 8 to 20 mpa ) . in the middle - third of the tooth , maximum displacement in the cervical portion was toward the buccal ( more than 500 microstrains ) . the maximum von mises stress distribution around the buccal bone ( surrounding isolated tooth ) was about 3.3 mpa ( with a range of 2 to 4 mpa ) . in the distobuccal area , 4 ) . in the second model ( isolated second premolar with both mesial and distal rests ) , the maximum von mises stress distribution in the buccal was 15 mpa ( with a range of 13 to 22 mpa ) and was concentrated at the cervical of the tooth . maximum stress distribution in the contact area of the i - bar clasp and cervical portion of the tooth was 30 mpa . stress distribution in the isolated second premolar tooth in model 1 from the distobuccal aspect . the overall length of this tooth was 24 mm , with 15 mm root length and 9 mm crown length . in the distal rest seat , the von mises stress distribution was 12.5 mpa ( which was much less distributed than the mesial rest seat with 17 mpa ) . in the distal proximal plate , tooth displacement in the cervical portion was 400 microstrains toward the buccal , with a mean value of 70 and the maximum of 1,150 microstrains . the von mises stress distribution around the bone ( surrounding the isolated tooth from the buccal aspect ) was 3 mpa , and the maximum stress was found in the distal alveolar bone crest and measured 5 mpa . in the third model ( the abutment tooth which was not isolated ) , the maximum von mises stress distribution was located in the cervical of the tooth ( in the buccal ) and also in the distal proximal plate . stress distribution in the buccocervical region was 7mpa ( with a range of 6.3 to 11.5 mpa ) . in the i - bar contact area , the stress distribution was about 15 mpa ; this value in the distal proximal plate was about 8 mpa ( with a range of 6.5 to 11 mpa ) . maximum stress distribution generated in the distal of the tooth was 15 mpa and was located around the contact borders of the proximal plate . maximum stress distribution in the borders of the mesial rest was also 15 mpa ( with a range of 6.8 to 11 mpa ) . in the tooth root , from the cervical one - third to the middle - third , the strain amount in the proximal plate of the second premolar was about 350 microstrains , and the maximum strain observed in this area was 700 microstrains . in this model , the von mises stress distribution in different areas of three models stress distribution in the cervicobuccal region of the bone around this abutment tooth was 2.1 mpa , and the maximum stress was observed in the distal area ( which was 5 mpa , with a wider range than lingual area ) . in this study , the numerical values of the von mises stress produced in different areas of mandibular second premolar abutments of rpds with different retainer designs were evaluated using 3d fem . during this evaluation , a static load was applied to underlying tissues in two different models with an isolated abutment and a model without a pier abutment . in consideration of the less bony support of mandibular teeth and preponderance of kennedy class 1 and 2 in rpd design generally , an identical situation for evaluating an abutment tooth can be obtained more easily through in vitro studies . fernandes et al , in a study using strain gauges stated that in vitro models of maxillary rpds are not suitable for accurate mechanical analysis . they further explained that in vitro studies can be useful only for simple determination of strain levels like our study . a fem is useful for assessing stress and strain levels in biomaterials and human tissues not easily measured by in vivo techniques . as photoelastic techniques provide two - dimensional and qualitative information , these stress patterns should be analyzed carefully . fernandes et al , in a study compared reflective photoelasticity and strain and concluded that reflective photoelasticity has important advantages such as validity , reliability and ease of use ; tests are not performed with a model ( i.e. like fem ) and the object itself is used for reflective photoelasticity . they explained that reflective photoelasticity has some limitations ; for example , it should be restricted to in vivo studies of the anterior teeth because it requires the accessibility of directed light under clinical situations . a further limitation is that reflective photoelasticity can only determine surface strain and direct loading of the coated surface should be avoided . reflective photoelasticity is better used in combination with other analytical techniques such as strain gauges , in order to completely analyze mechanical conditions of prosthetic appliances under load application . in our study , as direct loading was applied 3d fem was preferred over reflective photoelasticity in order to assess the abutment teeth . strain gauge techniques can be useful only for point stress analysis ; therefore , many gauges and difficult mathematical calculations are used . due to these limitations , we preferred to use 3d fem in this study . all the materials used in this study were considered to be linearly elastic , homogenous , and isotropic . wakabayashi et al stated that the force - movement relationship of a tooth can not be analyzed by a linear static model because this relationship is represented by a nonlinear curve . they stated that the linear static model should be used only in the studies measuring the difference of a tooth under limited range of load , like the current study . however , they stated that the validity and reliability of nonlinear fem in dentistry have not been adequately reported . mccartney in a clinical study explained that the clasp design has a direct influence on the direction and amount of transmitted force to abutment tooth . taylor et al indicated that tooth movement with a circumferential casting clasp is more than a rpi design , and that only with appropriately extended and adapted denture base , will the clasp changes be minimized . in different studies on mandibular bilateral distal extension partial dentures , physiologically adjusted rpds with an i - bar design and mesial occlusal rests have been recommended . aoda et al investigated the effect of three different retainer designs on loading of abutment teeth of a mandibular unilateral extension base rpd using 3d fem . in all designs , the load on the second premolar , which was adjacent to the region of the missing teeth , was high , suggesting that almost the entire load was supported by the distal abutment . akers clasps positioned over the first and second premolars produced the largest load on the abutment teeth . however , in our study i - bar design was used which is considered to cause less detrimental load on the distal abutment teeth , and the rpd frameworks were physiologically adjusted in all three models . he concluded that the rest position , rigidity and contour of the major connecter and the amount of extension base may have an effect on the abutment movement . in our study , maximum von mises stress distributions in all three models were located in the i - bar placed in the buccocervical region of abutments , distal proximal plate and the middle third of the root . the stress magnitude and distribution in the mesial rest seat was more than the distal rest seat . maximum stress magnitude in model 1 ( an isolated abutment with a mesial rest ) was 38.9 mpa and in model 2 ( the pier abutment with both mesial and distal rests ) was 36.7 mpa . the mean stress in model 1 was 2 mpa and the mean stress in model 2 was 1.6 mpa . finite element analysis ( fea ) is a numerical method in bioengineering and a valid method to predict and simulate different complex structures . by comparing these models , we found that the tooth movement in model 2 ( with both mesial and distal rests ) was less than the other two models . this finding may be a result of tooth engagement ( in this model ) with two rests and proximal plates . muraki et al , in a fea explained that mesially or distally positioned rest seats did not have a significant difference in abutment tooth movement . they stated that limiting horizontal rest movement in its seat ( through precise fitness ) may reduce abutment tooth movement in the horizontal dimension . under a similar assumption , we believe that less movement and strain in model 2 in our study ( with both mesial and distal rests ) may be justifiable . in our study , precise and firm fitness between all rests and rest seats in the three models was examined carefully . muraki et al reported maximum tooth movement toward the buccal , which is similar to the result of our study . they explained that by placing either a mesial or distal rest , maximum abutment tooth movement occurred in the mesial and buccal direction . the stress distribution in the distocervical alveolar bone crest of the isolated tooth ( in buccal ) was more than in the lingual surface ( which included sharp edges ) . considering possible tooth movement toward the mesial and buccal ( and the thinner buccal bone compared with the lingual bone ) , we believe that higher stress concentration in the buccal aspect is important . this issue is important to consider as it may result in increased bone resorption , pocket depth or tooth mobility . the prognosis of the abutment tooth of rpd depends on the amount of periodontal pocket depth and its mobility . base adaptation , which reduces as a result of time - dependent bone resorption , may itself increase force intensity applied to abutments . despite biomechanical factors , plaque accumulation and poor oral hygiene they observed that premolar tooth mobility increased only toward the buccal and there was no statistical difference in tooth mobility toward the lingual . they explained this finding to be the result of the thin buccal bone compared to the thick lingual bone . in a clinical study by tebrock et al tebrock et al explained that this result could be related to the lingual arm bracing . in our study , the stress concentration in all models was located on the buccal side of the abutment and in the middle - third of its root . this result may be due to the rotational movement of tooth around a center point in the root toward the buccal , which is similar to the studies by goodkind and tebrock et al . . however , according to the results of our study , tooth movement was toward the buccal , and strains generated in the bone of the abutment tooth did not seem to be more than the physiological tolerance threshold . in an in vitro study using strain gauge technique , el charkawi et al designed a mandibular bilateral distal - extension rpd with a resilient layer and circumferential clasps on the distal abutments ( the second premolars ) . they stated that strain gauge technique is an accurate , consistent and reproducible technique for in vitro studies of rpd designs . they used controlled load instead of static load by using an artificial oral environment ( artificial mouth ) . in all three axes of motion , the lingual strains were relatively higher . they stated that it might be the result of rpd design factors such as circumferential clasp pressure . their result contradicts the findings of our study . in our study , von mises stress in the buccal bone was more than in the lingual bone . of course , they used akers clasp on the distal abutments , which is contraindicated especially in combination with resilient - layer distal extension because it would exert extra detrimental forces to the distal abutment . despite the identical situations in all three models of the current study , greater stress distribution was found in models 1 and 2 with isolated abutments than in model 3 without an isolated tooth . the maximum von mises stress in models 1 and 2 was about 30 mpa . this result can be explained by the existence of more anterior neighboring teeth and the continuity of the dental arch in model 3 . the movements of rpds can occur in any of the three fundamental planes : horizontal , sagittal and frontal . the rpd movements include movements towards and away from the supporting ridge , and the mediolateral rotational movements that occur relative to the edentulous alveolar process . all these movements and resultant strains require intricate calculations that are too complicated for a single study . in our study ( like most other in vitro rpd articles either using fem or other methods ) , rpd movements that occurred toward the ridge were considered . the most dominant functional forces were applied around the molar regions , and these forces were considered in our study . in our study , we only considered forces applied vertically to the occlusal surfaces and toward tissues . the mastication cycle does have other forces with different directions ( for example away from tissues ) . we recommend further research and clinical studies in which an isolated tooth is used as an abutment for a fpd to compare it with other pier abutments either with or without a direct retainer of rpd . clasp assemblies with a different amount of proximal plate coverage can be designed in other studies . within the limitations of this study , the following conclusions were drawn : maximum stress distribution in all three models was at the cervicobuccal region of the tooth in the location of the i - bar retentive clasp , in distal guiding plane of the abutment , and the middle - third of the root.in model 2 , stress magnitude and distribution in the mesial rest seat was more than in the distal rest seat.the von mises stress distribution in model 1 was more than in model 2.the von mises stress distribution in models 1 and 2 with isolated tooth was much more than that in model 3.in models 1 and 2 , stress distribution was observed in the distocervical part of the alveolar bone crest surrounding the abutment.the von mises stress in the lingual bone was less distributed than in the buccal bone.maximum tooth movement in all three models was toward the buccal side . the tooth movement in model 2 with both mesial and distal rests was less than in models 1 and 3 . maximum stress distribution in all three models was at the cervicobuccal region of the tooth in the location of the i - bar retentive clasp , in distal guiding plane of the abutment , and the middle - third of the root . in model 2 , stress magnitude and distribution in the mesial rest seat the von mises stress distribution in models 1 and 2 with isolated tooth was much more than that in model 3 . in models 1 and 2 , stress distribution was observed in the distocervical part of the alveolar bone crest surrounding the abutment . the von mises stress in the lingual bone was less distributed than in the buccal bone . the tooth movement in model 2 with both mesial and distal rests was less than in models 1 and 3 .
objectives : this finite element method study aimed to compare the amount of stress on an isolated mandibular second premolar in two conventional reciprocal parallel interface designs of removable partial dentures ( rpds ) and the same rpd abutment tooth ( not isolated).materials and methods : a kennedy class 1 , modification 1 rpd framework was simulated on a 3d model of mandible with three different designs : an isolated tooth with a mesial rest , an isolated tooth with mesial and distal rests and an abutment with a mesial rest ( which was not isolated ) ; 26 n occlusal forces were exerted bilaterally on the first molar sites . stress on the abutment teeth was analyzed using cosmos works 2009 software.results:in all designs , the abutment tooth stress concentration was located in the buccal alveolar crest . in the first model , the von mises stress distribution in the contact area of i - bar clasp and cervical portion of the tooth was 19 mpa and the maximum stress was 30 mpa . in the second model , the maximum von mises stress distribution was 15 mpa in the cervical of the tooth . in the third model , the maximum von mises stress was located in the cervical of the tooth and the distal proximal plate.conclusion:we recommend using both mesial and distal rests on the distal abutment teeth of distal extension rpds . the abutment of an extension base rpd , which is not isolated in presence of its neighboring more anterior tooth , may have a better biomechanical prognosis .
You are an expert at summarizing long articles. Proceed to summarize the following text: it increases heart rate and cardiac mass , promotes cardiac arrhythmias , and elevates systolic blood pressure . it also shortens the ejection period and impairs diastolic function.1)2 ) rarely , pulmonary hypertension has been reported in association with hyperthyroidism.3)4 ) we report a patient with isolated pulmonary arterial hypertension accompanied by hyperthyroidism due to graves ' disease . a 54-year - old woman was admitted to the emergency room with dyspnea and generalized edema . physical examination revealed diaphoresis , jaundice , distended jugular veins , hepatomegaly , ascites , ankle edema , and decreased breath sounds in both lower lung fields . laboratory tests revealed leukocytosis without neutrophilia , normal cellular liver enzymes { aspartate aminotransferase ( ast)/alanine aminotransferase ( alt ) of 16/12 iu / l } , mild bilirubinaemia ( total bilirubin of 3.4 mg / dl ) , a prolonged prothrombin time of 17.7 seconds { international normalized ratio ( inr ) 1.7 } , and a normal partial prothrombin time of 28 seconds . the patient was given oxygen through nasal cannula and was administered intravenous furosemide for pulmonary congestion . she underwent a transthoracic echocardiogram , which revealed a dilated right ventricle , reduced right ventricular systolic function , a severely elevated systolic pulmonary arterial pressure of 80 mmhg , and moderate tricuspid valvular regurgitation . the left ventricular chamber was normal sized , with a reasonable ejection fraction of 0.75 ( fig . serum d - dimer was elevated at 2,460 ng / ml ( normal , 0 - 500 ng / ml ) . a chest computed tomogram obtained to rule out pulmonary embolism revealed no evidence of pulmonary embolism . a ventilation - perfusion pulmonary scan suggested a low probability of pulmonary embolism , as well . 2 ) . further laboratory tests revealed a decreased serum thyroid - stimulating hormone ( tsh ) level ( less than 0.08 uiu / ml ) , an increased serum free thyroxine ( ft4 ) level ( 4.45 ng / dl ; normal , 0.89 - 1.76 ng / dl ) , and an increased serum total triiodothyronine ( tt3 ) level ( 3.03 ng / ml ; normal , 0.60 - 1.81 ng / ml ) . a right heart catheterization performed the following day demonstrated severely elevated systolic / mean pulmonary artery hypertension of 80/60 mmhg . the pulmonary capillary wedge pressure was normal at 10 mmhg , without evidence of intracardiac shunt . anti - thyroid peroxidase antibodies were positive at 54.7 u / ml . the patient was confirmed to have idiopathic pulmonary artery hypertension and hyperthyroidism due to grave 's disease and was started on bosentan , amlodipine , and methimazole . during the following days , the patient 's clinical condition gradually improved , and her body weight returned to baseline . the patient was discharged in good clinical condition without signs or symptoms of heart failure . tsh was still low ( < 0.08 uiu / ml ) , and tt3 was slightly elevated ( 2.53 ng / ml ) , but ft4 was within the normal laboratory range ( 1.63 ng / dl ) . approximately four weeks later , a transthoracic echocardiogram revealed a normal sized right ventricle with good systolic function , improved systolic pulmonary artery pressure of 35 mmhg , and trivial tricuspid valvular regurgitation ( fig . tt3 improved ( 2.28 ng / ml ) , and ft4 remained in the normal range ( 1.34 ng / dl ) . we decided to discontinue bosentan , which is capable of masking the effect of the euthyroid state . after discontinuation of bosentan , the patient remained in good condition , without symptoms or signs of right - sided heart failure . many of the clinical manifestations of hyperthyroidism are attributable to the ability of thyroid hormones to alter cardiovascular hemodynamics . the usual manifestations occur secondary to high cardiac output causing left - sided heart failure . however , there is an increasing body of evidence suggesting that hyperthyroidism might cause reversible pulmonary arterial hypertension ( pah ) , resulting in right - sided heart failure . this phenomenon might be underdiagnosed , because pulmonary artery pressure usually returns to the normal range after a euthyroid state is achieved.5 ) the potential pathogenic mechanisms of hyperthyroidism - related pah remain unclear . it has been postulated that the pathogenesis of pah in hyperthyroid patients is similar to that of other autoimmune diseases related to pah.6 ) however , siu et al.7 ) reported that there was no significant difference in the prevalence of positive autoimmune antibodies between patients with pah and those without pah , or in the resolution of pah after successful antithyroid treatment regardless of the underlying etiology of hyperthyroidism .
we describe a 54-year - old woman with isolated pulmonary arterial hypertension accompanied by hyperthyroidism due to graves ' disease . her pulmonary artery hypertension resolved spontaneously after restoration of euthyroidism . this case suggests that hyperthyroidism should be considered a reversible cause of pulmonary arterial hypertension .
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Proceed to summarize the following text: precocious puberty is defined as children attaining puberty more than 2.5 to 3 standard deviations ( sd ) earlier than the median age , or before the age of eight years in girls and nine years in boys ; prevalence being 10 times higher in girls . the most common mechanism for progressive precocious puberty is early activation of the pulsatile gonadotropin - releasing hormone ( gnrh ) secretion ( central or gonadotropin - dependent precocious puberty ) , which results from hypothalamic tumours or lesions ; but in most cases ( approximately 90% ) it remains unexplained . the incidence of hypothalamic hamartomas ( hhs ) has increased since the introduction of mr imaging . the aetiology of this anomaly and the pathogenesis of its peculiar symptoms remain unclear , but recent electrophysiological , neuroimaging , and clinical studies have yielded important data that will be briefly reviewed in this article . no treatment is necessary in at least half of the cases of precocious puberty , as the gonadotropic axis is not activated and spontaneous regression of pubertal manifestations occur . in cases with progressive precocious puberty , they may present with adverse psychosocial outcomes , early menarche , and short adult stature , because of early epiphyseal fusion . we present a rare case of precocious puberty with clinical , laboratory , and radiological features supporting a central aetiology , presenting at an extremely early age ( six months ) . we also use this case to illustrate an approach to a case of isosexual ( central ) precocious puberty in girls . a sixteen - month - old girl was brought by her parents to the paediatric endocrinology outpatient department with a history of bleeding per vaginum since six months of age , with no previous history of genital trauma . she was the second child of the parents , with no positive history of consanguineous marriage . initially she had irregular cycles , which evolved into monthly regular cycles of three to four days for the next eight months . the developmental ( gross motor , fine motor , language , and social ) milestones were achieved normally . fresh blood was seen on the genitals , with active vaginal bleeding , and there was edema and erythema of the vaginal introitus , with no local erosions . she had normal haematological and biochemical profiles , however , the hormonal analysis revealed pubertal response of gonadotropins with luteinizing hormone ( lh ) of 2.20 mi/ ml ( n < 0.6 miu / ml ) , follicle - stimulating hormone ( fsh ) of 5.58 miu / ml ( n < 0.6 miu / ml ) , and estradiol ( e2 ) of 10.2 pg / ml ( n < 5 pg / ml ) , with normal thyroid functions . x - ray of the left wrist revealed bone age of 24 months [ figure 2 ] . tumor markers such as carcinoembryonic antigen ( cea ) , ca 19.9 , alpha - fetoprotein , and human chorionic gonadotropin ( hcg ) were negative . uterine volume on pelvic ultrasonography was 2.2 ml , with no evidence of ovarian cyst / tumour . magnetic resonance imaging ( mri ) of the brain ( plain [ figure 3a ] and contrast [ figure 3b ] ) revealed a well - defined , sessile hypothalamic mass , isointense to the gray matter on t1-weighted , and hyperintense on t2-weighted sequences , suggestive of hypothalamic hamartoma measuring 1.44 1.38 cm . the patient is being managed as a case of isosexual ( central ) precocious puberty , secondary to hypothalamic hamartoma , with monthly gnrh analogs ( triptorelin ) . the patient is under regular monthly clinical follow - up for seizures and secondary sexual characters , annual hormonal assays , bone age assessment , and mri brain , for changes in the hypothalamic hamartoma . the patient has shown good response on the two - year follow - up , with subsidence in size of the hypothalamic hamartoma , reversal of secondary sexual characters , and regression of monthly menstrual cycles . bilateral breast enlargement ( thelarche - stage b3 ) plain radiograph wrist showing bone age of approximately 24 months ( a ) plain mri brain ( t1w image ) revealing sessile , well - defined hypothalamic mass is intense to gray matter ( b ) mri brain , post contrast , revealing a hyper intense mass lesion suggestive of hypothalamic hamartoma , measuring 1.44 1.38 cm areas of uncertainty in evaluating cases of precocious puberty include , an appropriate age threshold for defining precocious puberty , approach to differentiate progressive from non - progressive forms , and causal mechanisms underlying idiopathic precocious puberty . to evaluate patients with suspected precocious puberty , internists should consider the following questions : is pubertal development really occurring outside the normal temporal range ? what is the etiology , and is there any risk of an intracranial lesion as the underlying mechanism ? is there any impairment in the child 's normal physical and psychosocial development secondary to the aberrations in the progression of pubertal development ? complete family history ( age at onset of puberty in first - grade relatives ) , any signs and symptoms suggesting possible central nervous system ( cns ) abnormality , such as , increase in head circumference , seizures ( in particular gelastic ) , visual impairment or headache should be the first step in evaluating the case . patients should be evaluated for high growth velocity , which may also precede the onset of pubertal manifestations and pubertal development , classified as per tanner staging . the extremely early age of onset ( six months ) of menarche and thelarche ( tanners stage b3 ) , with no positive family history in this patient , directed the diagnosis initially toward peripheral precocious puberty ( peripheral tumor or mccune - albright syndrome ) or the existence of an intracranial injury . the examination should comprise consideration for signs such as hyper pigmented skin lesions , suggestive of the mccune albright syndrome or neurofibromatosis , which were absent in our case . additional tests are recommended in patients with either tanner stage 3 or stage 2 , with increased growth velocity or symptoms and signs suggestive of cns dysfunction . on fulfilling the first of these above - mentioned criteria , we subjected this child to additional testing ( assessment of the bone age , hormonal analysis , and imaging ) . in precocious puberty , the bone age is generally greater than the chronological age , which can be assessed using a reference atlas such as by greulich and pyle . serum e2 levels in girls have low sensitivity in the diagnosis of precocious puberty due to its high variability . very high levels of e2 ( 100 pg / ml or 367 pmol / l ) generally indicate an ovarian cyst or tumour . the gold standard for determining precocious puberty is the gonadotropin assay post stimulation by gnrh or a gnrh - agonist , prior to starting therapy . peak lh levels of 5 8 miu / l suggest progressive central precocious puberty , with 100% specificity for a cut - off figure of 6 miu / l. unless levels of lh are clearly elevated , as in our case ( more than four times the ninety - fifth percentile ) , it is important to be careful while checking the levels of gonadotropin in children less than two to three years ( as the levels are generally higher in this age group ) . as the fsh levels have very little variation throughout pubertal development , its estimation is not very useful . in girls , ovarian cysts / tumors and uterine changes ( with 89% sensitivity and specificity for uterine volume 2.0 ml ) can be noticed on pelvic ultrasonography . this child had increased uterine volume , probably because of increased e2 ( twice the normal ) , but was found to have no evidence of any ovarian pathology . to establish whether a hypothalamic lesion is present , an mri of the brain should be performed in all cases of progressive central precocious puberty ( cpp ) . the prevalence of these hypothalamic lesions in cpp is lower in girls ( 8 to 33% ) when compared to boys ( 40 to 90% ) . the prevalence is much lower ( about 2% ) if puberty starts after the age of six years . our patient had a hypothalamic hamartoma measuring 1.44 cm 1.38 cm , which is the most common causes of cpp at this age . hypothalamic hamartomas are non - neoplastic , heterotopic nodules , resembling the normal gray matter of the hypothalamus . hhs occurs due to tissue displacement during the fifth or sixth week of gestation , when the ventral aspect of the neuraxis approaches the anterior tip of the end of the notochord . hh is associated with endocrinological , neurological symptoms , and symptoms due to the accompanying anomaly . neurological symptoms include gelastic seizures , pressure to laugh , visual impairment , focal seizures with secondary generalization , developmental delay , cognitive impairment , and behavioural disturbance . hh has been classified variably by arita et al . into parahypothalamic / intrathalamic and by bokyo et al . into various reported modalities include resection surgery , disconnection surgery ( disconnection from hypothalamus / corpus callosotomy ) , stereotactic radiation , radiofrequency thermocoagulation , and vagal nerve stimulation . internists evaluating a case of precocious puberty must take the decision on whether to provide treatment for girls , in particular , if onset of puberty is 8 years of age . the use of gnrh agonists continuously stimulates pituitary gonadotrophs , which further help in decreasing and desensitizing the release of lh , and to a lesser extent , fsh . to determine the desired effect of the therapy , a suppressed response of lh to gnrh or its agonist discontinuation of treatment at the age of 11 years aids in obtaining an optimal height and reappearance of pubertal manifestations . the european society for paediatrics endocrinology and its american counterpart , the lawson wilkins paediatrics endocrine society , are now preparing the consensus statement in relation to the use of gnrh agonists in children . we managed our case with inj tryptorelin ( gnrh agonist ) monthly depot injections , with remittance of pubertal changes ( clinical and hormonal ) . generally hypothalamic lesions , in association with precocious puberty , may lead to gonadotropin deficiency . thorough history taking and careful examination is required to determine the possible causes of precocious puberty , however , it is often vague . additional evaluation should include hormonal assays and bone age assessment ( e2 , lh , and tsh ) . if a randomly measured level of lh is in the pubertal range , an mri brain should be obtained . a pelvic ultrasound scan is required to rule out an ovarian tumour or cyst , mainly if the e2 level is elevated . a gnrh or gnrh - agonist stimulation test is the gold standard for diagnosing cpp , and is recommended to assess the activation of the gonadotropic axis , for predicting the progression of puberty . in case of progressive cpp , treatment with a depot gnrh agonist is suggested and is generally continued for 11 years , even though the best duration of therapy is undecided .
precocious puberty defined as an onset of puberty below eight years in girls and nine years in boys , has an incidence of approximately 1 / 5,000 1 / 10,000 subjects with a female / male ratio of 20 : 1 . it is etiologically classified broadly as central and peripheral . we present to you a case of isosexual ( central ) , precocious puberty in a 16-month - old girl , who was symptomatic since the age of six months , and was later , diagnosed to have hypothalamic hamartoma . it is one of the earliest case records ever in the medical literature of menarche , at an extremely early age ( six - month - old child ) secondary to a central cause .
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Proceed to summarize the following text: the diesters of phthalic acid ( also known as phthalates ) were introduced in the 1920s as plasticizers to soften plastics . because phthalates make products flexible and durable , they have been widely used in a variety of commercial and personal care products , including electronics , medical devices , toys , nail polishes , and perfumes . because phthalates can leach from these products , they are now ubiquitous in the environment . phthalates have been found in indoor dust , foodstuffs , indoor and outdoor air , and personal care products in the u.s . following exposure , phthalate diesters are metabolized to the corresponding monoesters in human bodies and excreted in urine . according to the national health and nutrition examination survey ( nhanes ) , 100% of the u.s . several studies have reported that phthalates are potential reproductive toxicants and endocrine disruptors , with a more pronounced effect in males . a negative association between semen volume and urinary concentrations of dibutyl phthalate ( dbp ) and di(2-ethylhexyl ) phthalate ( dehp ) and a positive association between sperm malformation and concentrations of dehp have been reported in males from china . similarly , a positive association was observed between sperm dna damage and urinary concentrations of phthalate metabolites in males from the u.s . studies also have shown that phthalate exposure can alter reproductive and thyroid hormone levels in males . laboratory animal studies have shown that dehp exposure can impair fertility in female mice . an epidemiological study in denmark reported an association between pregnancy loss and high exposure to dehp in females . several earlier studies have investigated the association of urine phthalate levels in pregnant women with outcomes for fertility and reproduction . these population - based studies provide evidence for widespread human exposure to phthalates and associate such exposures to impaired fertility and fecundity . although several mechanisms for reproductive toxicity of phthalates have been proposed , the health effects from low - level exposures are still not clearly understood . analysis of the effect biomarkers in human biospecimens , as well as exposure levels of phthalates , would provide further insights into the mode of toxicity of phthalates . oxidative stress occurs when a normal pro - oxidant to antioxidant ratio is imbalanced . this imbalance can play a key role in the pathogenesis of subfertility in both males and females . the adverse effects of oxidative stress on sperm quality and functions have been well documented . nevertheless , in females , the impact of oxidative stress on oocytes and reproductive functions remains unclear , given limited study . both lifestyle factors , such as dietary preferences , tobacco smoke , and alcohol consumption , and exposures to environmental chemicals can trigger oxidative stress . guanosines are susceptible to oxidation , and this reaction can lead to g : c t : a mutations that could have serious consequences . 8-ohdg is excreted in urine and , as such , provides an assessment of general oxidative stress throughout the body . an association between phthalate exposure and 8-ohdg has been reported in an occupationally exposed population ( plastic waste recycling workers ) in china . no previous studies have investigated the level of 8-ohdg in a cohort composed of couples who were planning pregnancy and an association of 8-ohdg with phthalate exposure . the human biospecimens collected for the longitudinal investigation of fertility and the environment ( life ) study , designed to examine the relationship between environmental chemical exposures , lifestyle , and couples fecundity and fertility , provided an opportunity to assess the association between phthalate exposure and 8-ohdg levels in urine . in a previous study , we showed that select phthalates were associated with diminished couple fecundity , especially in males , as measured by a longer time - to - pregnancy , in this longitudinally followed cohort recruited prior to conception . in the present study , 14 phthalate metabolites were quantified in 894 urine samples collected from 469 couples from michigan and texas during 20052009 . free urinary 8-ohdg , a marker of dna oxidative damage , also was determined . the objectives of this study were to determine exposure doses , concentrations , and profiles of phthalate metabolites in couples planning pregnancy and to examine the relationship between urinary phthalate levels and 8-ohdg . the life study used a prospective cohort design suitable for following couples across sensitive windows of human reproduction and development . in the study , couples who planned to become pregnant were recruited from michigan and texas between 2005 and 2009 . further details of the recruitment of the population , study design , and baseline demographic information have been described previously . the selection criteria were as follows : females between 18 and 44 years of age and males 18 years of age , partners in a committed relationship , neither partner being medically / surgically sterile , the female s menstrual cycle as between 21 and 42 days , no hormonal birth control injections used in the previous 12 months , and both partners able to communicate in english or spanish . in this study , baseline urine samples ( n = 849 ) collected from 469 couples at the initial stages of recruitment urine samples were transported to our laboratory on dry ice and stored at 80 c until analysis . based on previous stability studies , it was determined that the method used for transportation and storage of urine did not affect the levels of urinary phthalates and 8-ohdg . phthalate metabolites in urine samples were extracted after enzymatic deconjugation , followed by solid - phase extraction ( spe ) , using an isotope diluted method as described previously . briefly , 0.5 ml of urine sample was buffered with 200 l of ammonium acetate ( ph = 4.5 ) , and 50 l of -glucuronidase ( 2 l / ml ; from helix pomatia ) , 25 ng each of c - labeled phthalate metabolite standards ( as internal standards ) , and 0.5 ml of milli - q water were added . a rapidtrace spe workstation ( caliper life sciences , hopkinton , ma ) was used for the extraction of target analytes . an abs elut - nexus ( varian , walnut creek , ca , usa ; 60 mg/3 ml ) spe cartridge was conditioned with 1.5 ml of acetonitrile and 1.2 ml of phosphate buffer ( ph = 2 ) . urine samples were diluted with 1 ml of phosphate buffer and loaded onto the cartridge at a rate of 0.5 ml / min . the cartridge was rinsed with 2.0 ml of formic acid ( 0.1 m ) and 1.2 ml of milli - q water and dried under nitrogen for 5 min . target compounds were eluted from the cartridge with 1.2 ml of acetonitrile , followed by 1.1 ml of ethyl acetate . the eluate was concentrated under a gentle stream of nitrogen to near dryness , and 0.5 ml of acetonitrile : milli - q water ( 1:9 ) was added . 0.2 ml of urine was diluted 5-fold with milli - q water , and 10 ng of labeled internal standard ( n5 - 8-hydroxy-2-deoxyguanosine ; 99% , cambridge isotope laboratories , andover , ma , usa ) was added and analyzed by multiple reaction monitoring ( mrm ) in the positive ionization mode by lc an api 2000 electrospray triple quadrupole mass spectrometer ( esi - ms / ms ; applied biosystems , foster city , ca ) equipped with an agilent 1100 series high - performance liquid chromatograph ( hplc ) ( agilent technologies inc . , santa clara , ca ) was used for the measurement of phthalate metabolites . a total of 14 phthalate metabolites , mono(3-carboxypropyl ) phthalate ( mcpp ) , monomethyl phthalate ( mmp ) , monoethyl phthalate(mep ) , mono(2-isobutyl phthalate ( mibp ) , mbp , mono(2-ethyl-5-carboxypentyl ) phthalate ( mecpp ) , mono-[(2-carboxymethyl)hexyl ] phthalate ( mcmhp ) , mono(2-ethyl-5-oxohexyl ) phthalate ( meohp ) , mono(2-ethyl-5-hydroxyhexyl ) phthalate ( mehhp ) , monocyclohexyl phthalate ( mchp ) , monobenzyl phthalate ( mbzp ) , mehp , monoisononyl phthalate ( mnp ) , and monooctyl phthalate ( mop ) , were analyzed . seven isotopically labeled phthalate metabolites ( c4-mmp , c4-mep , c4-mbp , c4-mecpp , c4-mehp , c4-mbzp , and d4-mibp ) were used as internal standards . chromatographic separation was achieved using a betasil c18 column ( thermo electron , bellefonte , pa ; 100 mm 2.1 mm , 5 m ) . the mobile phase was 0.1% acetic acid in milli - q water ( a ) and 0.1% acetic acid in acetonitrile ( b ) at a flow rate of 300 l / min . the limits of quantification ( loq ) of phthalate metabolites varied from 0.1 to 0.5 ng / ml . 8-ohdg was analyzed using the same instrumental parameters as presented above , and quantification was achieved by isotope dilution . the loq for 8-ohdg was 0.2 ng / ml . for each batch of 60 samples , four method blanks , a spiked blank , and a pair of matrix - spiked sample / duplicates were processed . the qa / qc results are provided in table s1 ( supporting information ) . the average recoveries of c - labeled internal standards in all samples were between 52% and 68% . the reported concentrations in samples were corrected for the recoveries of the internal standards . the coefficients of variation of duplicate analysis of samples were between 5% and 36% . trace levels of mep , mbp , mibp , mecpp , and mehp were found in procedural blanks , and these levels ( average values of the four blanks in each batch ) were subtracted from sample values . if the final concentration was a negative value ( due to subtraction of background levels ) , half of the lowest concentration found for each compound was used in data analysis . the sum concentrations of five metabolites of dehp ( mehp , mecpp , mcmhp , mehhp , and meohp ) and the sum of all 14 phthalate metabolites are referred to as 5mehp and 14phthalates , respectively . . a comparison of concentrations between several subgroups was performed using a nonparametric test ( kruskal wallis h or wilcoxon signed - rank test ) . the association between 8-ohdg and phthalate metabolite concentrations was tested by partial correlations ( age , bmi , smoking habit , and race as controlling variables ) . the relationship of phthalate concentrations between paired males and females was examined by linear regression . phthalate metabolites were found with detection frequencies of > 90% for mep , mcpp , mbp , mibp , and metabolites of dehp ( except for mehp ) and mbzp , 40% for mmp , and 50% for mehp . mchp , mnp , and mop were found in < 5% of the urine samples analyzed ; we did not include these compounds in our further discussions . concentrations and profiles of phthalate metabolites in urine from the study population are presented in table 1 . mep and the metabolites of dehp were the most abundant compounds , accounting for 90% of the total concentrations in urine , followed by mbzp , mbp , mibp , and mmp . the median concentrations of mmp , mep , mcpp , mbp , mibp , mbzp , 5mehp , and 14phthalates were 0.48 , 85.2 , 4.50 , 7.66 , 4.36 , 3.80 , 54.8 , and 249 g / g creatinine , respectively ( n = 894 ) ( table 1 ) . the mean concentrations in all samples were 1.3/1.76 ( g / g creatinine / ng / ml unadjusted ) , 233/259 , 10.8/13.8 , 14.9/16.9 , 6.34/7.33 , 6.62/8.59 , 164/190 , and 437/497 for mmp , mep , mcpp , mbp , mibp , mbzp , 5mehp , and 14phthalates , respectively . among the five metabolites of dehp , mecpp , mcmhp , and mehhp were the predominant compounds , with median concentrations of 15.8 , 13.9 , and 12.3 g / g creatinine , respectively . our values were similar to the concentrations reported for phthalate metabolites in urine from the general u.s . population in 20032004 by nhanes ( median concentrations for mep , mbp , mibp , mbzp , and dehp metabolites were 177 , 17.7 , 3.33 , 10.5 , and 54.2 g / g creatinine ) , with the exception of mep , mbp , and mbzp , which were 2 times lower in our study . nd : concentrations were lower than 0.01 g / g creatinine . * = p < 0.05 . * significantly higher concentrations of phthalate metabolites , except for mmp , were found in females as compared to males ( table 1 ) . especially , concentrations of mbp were 2 times higher in females than in males ( median value ) . in addition , between paired samples of 424 couples ( wilcoxon signed - rank test ) , concentrations of phthalate metabolites were significantly higher in females as compared to males , except for 5mehp . these results were similar to those reported in previous studies ; higher levels of phthalates in females were explained by the use of personal care products , such as nail polishes , skin toners , and liquid foundations , which contain high concentrations of phthalates . our previous study showed that phthalate diesters such as dep were frequently found in leave - on personal care products , by up to 0.4% of the product weight . lack of differences between males and females for 5mehp levels can be explained by similar dietary habits within a family , as diet is the major source of human exposure to dehp . further , linear relationships were found for mep ( r = 0.11 , linear regression model value ) , mibp ( r = 0.21 ) , mbzp ( r = 0.22 ) , and 5mehp ( r = 0.11 ) between paired female and male partners ( figure s1 , supporting information ) , which indicated coexposures to phthalates by couples in each family . concentrations of mbp and mibp were significantly higher in individuals with lower bmis ( < 25.0 ) ( 8.77 and 4.66 g / g creatinine , median ) than those with higher bmis ( 25.0 ) . however , when the urinary concentrations were not normalized for creatinine , no relationship was found between bmi and mbp / mibp concentrations ( figure s2 , supporting information ) . this difference can be explained by the excretion of higher levels of creatinine by individuals with higher bmis ( median concentrations of 73 , 100 , and 135 g / g for the three bmi groups ) . in addition , unadjusted concentrations of mep , mcpp , mbzp , 5mehp , and 14phthalates were significantly higher in individuals with higher bmis ( table s2 , supporting information ) for both males and females . these results were similar to those reported previously , which showed a positive relationship between the concentrations of mbzp , mep , mbp , and dehp metabolites and waist circumference or bmi in males ( creatinine adjusted ) and between the concentrations of mep and obesity in children ( both creatinine adjusted and unadjusted ) . these results further suggest positive associations between phthalate exposure and overweight or obesity . among different ethnic groups studied , significantly higher concentrations of 5mehp were found for caucasians ( 59.8 g / g creatinine ) and asians ( 54.6 g / g creatinine ) than for other ethnic groups ; concentrations of mep and mbzp were significantly higher for african americans ( 183 and 4.62 g / g creatinine ) than for other races , and concentrations of mibp and mbp were significantly higher for asians ( 13.6 and 7.09 g / g creatinine ) than for other races . it has been reported that the concentrations of mep for non - hispanic blacks and mexican americans were significantly higher than those for non - hispanic whites ( 254 , 212 , and 165 g / g creatinine , respectively ) . in our previous study , we reported elevated concentrations of mbp and mibp in urine from asians . these results suggest differences in sources of exposure to phthalates among various ethnic groups , which may be related to lifestyle and cultural practices . significantly higher concentrations of mcpp and 5mehp were found in nonsmokers ( 4.61 and 56.7 g / g creatinine ) than in smokers ( 3.54 and 42.2 g / g creatinine ) . the mean and median concentrations of 8-ohdg in urine were 6.02 and 3.13 g / g creatinine , respectively ; the corresponding unadjusted mean and median concentrations were 4.21 and 3.30 ng / ml , respectively . in leaner individuals ( i.e. , low bmi ) , significantly higher levels of 8-ohdg were found in urine from females than from males and from asians ( table 1 ) . previous studies have reported higher concentrations of 8-ohdg in females than in males . as a biomarker of oxidative dna damage , urinary levels of 8-ohdg can be affected by aging and cigarette smoking . a significantly higher concentration of 8-ohdg was found in the blood of people exposed to environmental tobacco smoke . nevertheless , no significant differences were observed among various age groups or between smokers and nonsmokers in the present study . this observation may be explained by the narrow age range ( reproductive age ) or limited number of smokers ( n = 115 ) in this study . however , a trend of increasing levels of 8-ohdg with age was found in females , with respective median values of 3.19 , 3.99 . 3.76 , and 4.26 g / g for the age groups 25 , 2630 , 3135 , and 36 years ( table 1 ) . the relationship between urinary concentrations of 8-ohdg and phthalate metabolites was investigated ( table 2 ) . for females , significant relationships were found between 8-ohdg and mep , mehp , 5mehp , and 14phthalates . for males , significant associations were found between 8-ohdg and mibp and mehp concentrations . a recent study showed that occupational exposure to dehp among male workers in plastic waste recycling industries increased urinary levels of 8-ohdg . laboratory rat studies showed elevated levels of 8-ohdg in urine following exposure to dbp or butyl benzyl phthalate ( bzbp ) and mbp ; nevertheless , a significant positive relationship was only found between urinary 8-ohdg and mehp or mibp levels in our study . however , it should be noted that statistically significant relationships were observed between 8-ohdg and urinary mmp , mep , mbp , mbzp , mehp , 5mehp , and 14phthalate concentrations in females , and urinary mmp , mcpp , mibp , mbzp , and 14phthalates in males , when the levels were not normalized for creatinine ( table s3 , supporting information ) . our results suggested that exposure to phthalates , especially dep , dbp , diisobutyl phthalate ( dibp ) and dehp , increases oxidative stress . oxidative stress can cause a decline in the levels of critical cell cycle factors that are related to reproduction and fertility and directly damage multiple intracellular components of the oocyte , such as lipids , proteins , and dna . a recent study showed that high concentrations of 8-ohdg in urine were significantly correlated with lower sperm count , lower sperm mobility , and abnormal sperm morphology . we reported earlier that select phthalate exposures ( mep , mbp , and mbzp ) in males from this study were negatively associated with time to achieve pregnancy . although several lifestyle factors , including dietary preferences , can contribute to oxidative stress that would affect fertility and fecundity , this study indicated the possibility that phthalate exposures can result in oxidative stress , which eventually effects reproductive performance in couples . for the estimation of human exposure doses of phthalates , a back - calculation method that involves extrapolation of concentrations determined in urine was used . based on the measured concentrations of urinary phthalate metabolites ( both unadjusted and creatinine adjusted ) , daily intake ( dis ) doses of phthalates were estimated by following equations as employed by koch et al.12where di is the daily intake of phthalates ( mg / kg / day ) , cuc ( g / g creatinine ) and cu ( g / l ) are creatinine adjusted and unadjusted concentration of urinary phthalate metabolite , fcre is the creatinine excretion rate ( mg / day ) , vu is the daily urine volume excreted ( l / d ) , bw is the body weight ( kg ) , fue is the molar fraction of the excreted metabolite in relation to the ingested parent phthalate molecule , and mwd and mwm are the molar weights of the parent phthalate and the corresponding metabolite . for fcre , values of 1.0 g / day for females and 1.7 g / day for males were applied , as reported previously ; for vu , 2 l / d was applied as a conservative estimate , and for fue , 0.69 for mmp , mep , mbp , and mibp , 0.73 for mbzp , 0.23 for mehhp , and 0.15 for meohp were applied . the highest estimated di values were found for dep , followed by dehp , dbp , dibp , bzbp , and dimethyl phthalate ( dmp ) ( figure 1 ) . the median dis of phthalates calculated from creatinine - adjusted urinary concentrations were slightly lower than the values calculated from unadjusted urinary data , and the values were 0.01 , 2.25 , 0.22 , 0.13 , 0.10 , and 1.0 g / kg - bw / day for dmp , dep , dbp , dibp , bzbp , and dehp , respectively ( table 3 ) . the mean di values were 2 to 3 times higher than the median di values for all phthalates and were 0.04 , 6.31 , 0.47 , 0.19 , 0.18 , and 4 g / kg - bw / day for dmp , dep , dbp , dibp , bzbp , and dehp , respectively . these estimated di values were lower than the oral reference doses ( rfds ) recommended by the u.s . epa , which are 100 , 200 , 800 , and 20 g / kg - bw / day , for dbp , bzbp , dep , and dehp , respectively . nevertheless , the dis estimated for dehp for 39 individuals ( 4.4% of the population ) ( calculated from mehhp ) were above the rfd of 20 g / kg - bw / day . estimated daily intake ( g / kg - bw / day ) of phthalates for males and females ( from the urinary metabolite concentrations ) in couples in texas and michigan planning pregnancy , 20052009 . horizontal lines represent the 10th , 50th , and 90th percentiles , and the boxes represent the 25th and 75th percentiles . dehp1 and dehp2 are di of dehp estimated from the concentrations of mehhp and meohp , respectively . median value : cre , calculated from creatinine adjusted phthalate concentration ; unadj , calculated from concentration values without creatinine adjustment . * = p < 0.05 , * * = p < 0.01 . for the u.s . general population , it was reported that the dis for dep and dehp were 5.411.4 and 2.16.5 g / kg - bw / day ( geometric mean ) , respectively . in another study , the median dis of dep , bzbp , dbp , dibp , and dehp for pregnant women in the u.s . were estimated to be 6.64 , 0.50 , 0.84 , 0.12 , and 1.32 g / kg - bw / day , respectively . for the general population in europe , the median dis of dep , dbp , bzbp , and dehp were reported to be 1.43/1.15 , 3.53/3.61 , 0.27/0.31 , and 2.54/2.85 g / kg - bw / day for females / males , respectively . for the population in china , the estimated median dis for dmp , dep , dbp , and dehp were 0.6 , 1.1 , 8.5 , and 2 g / kg - bw / day , respectively . the dis of phthalates estimated for the life study cohort are similar to those reported in other studies , but the dis estimated for dehp for 39 individuals ( 4.4% of the population ) ( calculated from mehhp ) were above the rfd of 20 g / kg - bw / day . no significant difference in the dis of phthalates was found among the four age groups or between smoking habits , except for significantly higher intakes of dehp among nonsmokers ( table 3 ) . the estimated dis of dmp , dep , and dbp were higher for texas than for michigan couples , the dis of dbp and bzbp were higher for females than males , and the dis of dbp , dibp , bzbp , and dehp were higher in leaner individuals ( low bmi ) than in obese individuals . significant positive relationships were found between the estimated dis of dmp , dep , dbp , and bzbp for females and dmp , dibp , and bzbp for males ( calculated from unadjusted urinary concentrations ) and 8-ohdg concentrations in urine . the results further indicate that exposure to phthalates induces oxidative stress , which is a factor associated with reduced fertility and fecundity .
oxidative stress has been recognized as one of the most important contributors to infertility in both males and females . exposure to many environmental chemicals , such as phthalates , has been shown to induce oxidative stress . in a longitudinal study designed to assess exposure to environmental chemicals and fecundity in couples who were planning pregnancy , 894 urine samples were collected from 469 couples from michigan and texas during 20052009 . the concentrations of 14 phthalate metabolites and a marker of oxidative stress , 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) , were determined in these samples . concentrations , profiles , and estimated daily intakes ( dis ) of phthalates were positively associated with 8-ohdg . the median concentrations of monomethyl phthalate ( mmp ) , monoethyl phthalate ( mep ) , mono(3-carboxypropyl ) phthalate ( mcpp ) , mono - n - butyl phthalate ( mbp ) , mono(2-isobutyl ) phthalate ( mibp ) , monobenzyl phthalate ( mbzp ) , 5mehp ( sum of five metabolites of di(2-ethylhexyl ) phthalate ( dehp ) ) and 14phthalates ( sum of 14 urinary phthalate metabolites ) were 0.48 , 85.2 , 4.50 , 7.66 , 4.36 , 3.80 , 54.8 , and 249 g / g creatinine , respectively . the estimated di values for dehp in 39 individuals were above the u.s . environmental protection agency s ( epa ) reference dose ( rfd ) of 20 g / kg - bw / day . the mean and median concentrations of 8-ohdg were 6.02 and 3.13 g / g creatinine , respectively , which were significantly higher in females than in males . statistically significant associations were found between 8-ohdg and urinary concentrations of mep , and 5mehp for females . similarly , a significant association was found between 8-ohdg and dis estimated for select phthalates . our results suggested that phthalate exposure increases oxidative stress , which can be a mechanism for the diminished fertility observed in couples who were highly exposed to select phthalates .
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Proceed to summarize the following text: regeneration of the lost periodontium as a consequence of periodontal diseases yet unachieved by surgery and/or any other means to its previously existing normal physiologic level is one of the main goals of periodontal therapy . conventional periodontal treatments , such as scaling and root planing , are highly effective at repairing disease - related defects and halting the progression of periodontitis but do little to promote regeneration of the lost periodontium . regenerative periodontal surgery using both tissue engineering and in situ tissue regeneration is modified continuously to modulate regenerative processes , though they may have variable success rate and efficacy in human jaw bones . all the currently available bone graft materials neither cure all nor cure up to 100% of any periodontal defect . ceramics , collagen , and polymers are the recently used alloplastic materials ; they can be either bioactive or bioinert . bioactive ceramics include hydroxyapatite ( ha ) , fluorapatite , bioactive glass ( bg ) , and tricalcium phosphate . tricalcium phosphate ( tcp ) has unpredictable high rate of bioresorption and incapable to induce periodontal regeneration . synthetic ha is a biocompatible , nontoxic , slowly resorbing , osteoconductive , osteophillic material and has close structural and chemical resemblance to bone mineral , but not identical . bioactive glass can develop a chemical bond with living hard tissues through the development of a surface layer of carbonated hydroxyapatite . when bg is exposed to tissue fluid , it is covered by silica rich gel and top of which calcium phosphate rich layer is formed that promotes absorption and concentration of osteoblast cells to form an extra cellular matrix and mineralization . kim et al . reported that bg guides and promotes osteogenesis and allows rapid bone formation . fetner et al . had studied comparison of tcp , ha , bg with an unimplanted control and found that bioactive glass ( perioglas ) produced significant bone and cementum repairs . on the other hand , therefore , the present study was aimed to evaluate and compare clinically and radiographically the effect of ha , bg , and bg - ha composite bone graft particles in the treatment of human infrabony periodontal defects . this study was carried out in the department of periodontics , dr . r. ahmed dental college , kolkata , in association with the central glass and ceramic research institute , kolkata . % ] that were developed ( by co - precipitation technique and conventional glass melting technique ) and characterized earlier both in vitro and in vivo were used for the present study . bg - ha composite graft was developed by milling the scaffold made up of 50% bg and 50% ha powder at 820c . twenty two systemically healthy patients ( 12 male and 10 female ) , aged from 30 to 56 years ( mean age , 43 years ) were selected for this study . radiographic examination revealed 28 three walled infrabony defects , and out of the 22 patients , 4 patients demonstrated multiple angular periodontal defects [ figure 1f ] , and were treated by open flap debridement ( ofd ) alone in one defect and ofd with identical synthetic bone graft ( ha or bg or bg - ha ) in the others . the informed consent was obtained from all the patients and the study was approved by the institutional ethical committee of dr . the infrabony defect sites were divided equally into four groups ( seven in each group ) - group i ( open flap debridement [ ofd ] was done alone ) , group ii ( ofd with ha ) , group iii ( ofd with bg ) , and group iv ( ofd with bg [ 50% ] ha [ 50% ] composite bone graft filling sites ) . patients selected for the study fulfilled the following inclusion criteria ; at least one clinical evidence of an infrabony defect , radiographic evidence of bone loss , systemically healthy patients , no history of long term antibiotics or other medications usage , and no history of invasive periodontal therapy for the past six months at the recipient site . acrylic stents with one prepared vertical groove were used as fixed reference position ( i.e. junction of vertical groove and lower border of the stent ) and for proper alignment of graduated periodontal probe university of north carolina ( unc-15 ) in subsequent measurements . ( a ) recording of relative attachment gain using acrylic stent , ( b , f ) infrabony defects after curettage and debridement ; infrabony defects implanted with ( c , i ) bioactive glass , ( d , j , k ) hydroxyapatite and ( e , g , h ) bg - ha composite the following clinical parameters were recorded to the nearest millimeter with the help of a unc-15 probe for each surgical site during surgery ( baseline ) and six months post - operatively . all the measurements as well as clinical procedures were conducted by a single examiner to exclude inter - examiner variation . soft tissue parameters were evaluated by plaque index ( pi ) , gingival index ( gi ) , probing pocket depth ( ppd ) , and relative attachment level ( ral ) . ral is the distance from a fixed reference point to base of the periodontal pocket [ figure 1a ] . hard tissue parameters were evaluated by depth of the defect ( dod ) and radiographic bone fill as seen in intra oral peri - apical ( iopa ) radiograph . dod is the distance from the fixed reference point to the deepest part of the defect that was measured at baseline by bone sounding and via surgical entry and after six months post - operatively through bone sounding only . the radiographic bone fill was assessed comparing six months post - operative radiographs with the baseline radiographs which were taken prior to the surgery . each patient was prepared for surgery with oral hygiene instructions , scaling and root planing at least three months prior to surgery , adjunctive chemical plaque control , and occlusal adjustment was performed , whenever necessary . patients were re - evaluated to assess clinical parameters and plaque control four weeks after initial therapy . after extra and intra oral disinfection and local anesthesia ( 2% lignocaine ) application , crevicular incisions were given and the flaps were elevated . depth of the defect from the fixed reference point ( apical end of the stent ) to the deepest point of the defect was measured for baseline reference . after thorough debridement and root planning [ figure 1b ] , an adequate quantity of the sterile synthetic bone graft particles were mixed with a few drops of saline in a sterile dappen dish . , a 3 - 0 non resorbable silk suture was passed through the buccal and lingual papillae and the suture was left loose to prevent removal of the graft particles by the passage of the needle as well as the suture material [ figure 1d ] . after placement of the graft [ figure 1c k ] , silk sutures were used to approximate the lingual and buccal flaps using interrupted sutures [ figure 1l ] and conventional periodontal dressing was given . in seven defect sites , no synthetic bone graft particles were implanted ( group i ) . asepsis and gentle tissue handling , which are important considerations for regenerative procedures , were strictly followed in each surgery . patients were prescribed 0.2% chlorhexidine twice daily for two weeks , oral antimicrobials ( 500 mg amoxicillin thrice daily 7 days ) , analgesic ( 400 mg ibuprofen thrice daily 3 days ) , antacids ( ranitidine 150 mg twice daily 3 days ) , and routine post surgical instructions were explained . patients were recalled after one week for suture removal and six months after surgery . at each of the recall visits oral hygiene was checked and oral hygiene instructions were reinforced . statistical analysis was employed to compare the study results using a software program . for each outcome measurement , at least 95% confidence limit was estimated for change of all observations from baseline to six months post - operatively . inter - group comparisons of changes in soft tissue and hard tissue parameters from baseline to six months post surgery were made between group i ( unimplanted control ) , group ii ( ha ) , group iii ( bg ) , and group iv ( bg - ha composite ) by independent sample t test and within the respective groups themselves ( intra - group ) through a paired student statistical analysis was employed to compare the study results using a software program . for each outcome measurement , at least 95% confidence limit was estimated for change of all observations from baseline to six months post - operatively . inter - group comparisons of changes in soft tissue and hard tissue parameters from baseline to six months post surgery were made between group i ( unimplanted control ) , group ii ( ha ) , group iii ( bg ) , and group iv ( bg - ha composite ) by independent sample t test and within the respective groups themselves ( intra - group ) through a paired student healing was uneventful in all the groups . in the intra - group comparison ( paired student t test ) , changes in soft tissue and hard tissue parameters of periodontal defects from baseline to six months post surgery for group i ( unimplanted control ) , group ii ( ha ) , group iii ( bg ) , and group iv ( bg - ha composite ) are presented in table 1 , and the inter - group comparison ( independent sample t test ) are presented in table 2 . no sites in group i , group ii , group iii , and group iv displayed 100% vertical defect resolution . mean plaque index scores for all the four groups were 1 at baseline , one week and 6 months after surgery ( no statistically significant differences - p>0.05 ) . mean gingival index scores at baseline and after six months of surgery for all the four groups were shown in table 1 ( ranged from 0 to 1 ) . paired t - value of gingival index ( gi ) scores at baseline , 1 week and 6 months after surgery in the four groups showed no statistically significant differences ( p>0.05 ) . the ppd values for the grafted sites ranged from 6 to 10 mm and in the control sites ranged from 6 to 8 mm at baseline . at six months , probing pocket depth values for the ha sites ranged from 4 to 6 mm , 2 to 4 mm for the bioactive glass group , 3 to 4 mm for ha - bg composite bone graft group , and 3 to 5 mm in the control group . the mean of ppd values for all the groups at baseline and six months after surgery were presented in table 1 . all the groups resulted in significant reductions in ppd ( p<0.01 ) . when comparison was done between the four groups bioactive glass group showed statistically significant reductions in ppd at 5% level compared to gr - i and gr - ii [ table 2 ] . the ral values at baseline ranged from 10 mm to 14 mm for ha group , 11 to 16 mm for bioactive glass group , 11 to 15 mm for ha - bg composite group and 10 to 12 mm in control group . at six months , ral values for the ha sites ranged from 8 to 9 mm and 7 to 9 mm for all the remaining groups . the mean of ral values for all the groups at baseline and 6 months after surgery were presented in table 1 . all the four groups resulted in significant gain in relative attachment level ( p<0.01 ) . when comparison was done between the four groups , ha - bg composite group showed statistically significant gain in ral at 5% level compared to control group only [ table 2 ] . the depth of defect ( dod ) values at baseline ranged from 11 to 15 mm for ha , 12 to 16 mm for bioactive glass group , 12 to 16 mm for ha - bg composite group , and 11 to 14 mm in control group . at six months , dod values for the ha sites ranged from 9 to 11 mm , 8 to 10 mm for the bioactive glass group , 8 to 12 mm for ha - bg composite group , and 9 to 10 mm in the control group . the mean of dod values for all the groups at baseline and 6 months after surgery were presented in table 1 . all the four groups showed statistically significant ( p<0.01 ) amount of defect fill from baseline to six months post operatively . the bioactive glass and ha - bg composite group resulted in a greater defect fill as compared to the ha and control group [ table 2 ] , but inter group comparison between the ha - bg composite and bioactive glass group showed no statistically significant difference in defect fill [ table 2 ] . intra - group comparisons of mean and sd of soft tissue and hard tissue parameters ( mm ) inter group comparisons ( independent sample t - test ) of different parameters studied in figure 2 , the representative iopa x - ray image of the healing site of group i , group ii , group iii , and group iv , a1 , a2 , a3 , and a4 show pre - operative and b1 , b2 , b3 , and b4 show six months post operative pictures of the respective groups of healing of infrabony periodontal defects . in the post operative pictures , b3 ( group iii ) and b4 ( group iv ) showed increased angular defect fill compared to b1 ( group i ) and b2 ( group ii ) in respect of their pre - operative view ( a3 , a4 and a1 , a2 ) . pre - operative ( a ) and six months post - operative ( b ) view of group i , group ii , group iii , and group iv ( periodontal defects ) , respectively a multitude of new materials have been used for promoting periodontal regeneration in infrabony defects where bioactive glass ( bg ) has gained momentum in the recent years . long - term success with regenerative procedures statistically insignificant differences ( p>0.01 ) in pi scores throughout the study period in the four groups [ table 1 ] suggesting that minimum amount of plaque did not interfere with regenerative process of any particular group and all the patients maintained their oral hygiene properly throughout the study periods , though insignificant deterioration in plaque scores might be found sometimes . similarly , statistically insignificant differences ( p>0.01 ) in gi scores in all the four groups [ table 1 ] suggesting that all the synthetic graft materials used were biocompatible , non toxic , didnt elicit any inflammatory and/or foreign body responses to the tissues , and no apparent retardation of normal bone mineralization process that claimed to be primary requisites for graft materials . intra - group comparisons of changes in ppd , ral , and depth of the defect [ table 1 ] from baseline to six months post surgery for all four groups showed statistically significant improvement ( p<0.01 ) suggesting that both ofd and ofd plus synthetic bone graft particles were effective in the treatment of infra bony periodontal defects . in inter - group comparisons [ table 2 ] , there was significant improvement ( p<0.05 ) in the probing pocket depth in group - iii ( bg ) as compared to the group - i ( control ) and group - ii ( ha ) suggesting that bg composition ( 43 - 44 wt% sio2 ) used in this study was below 52% by weight of sio2 that could form a bond with soft tissue collagen . it acted as a biologic membrane retarding epithelial downgrowth and appeared to facilitates growth and repair of soft connective tissues . but statistically insignificant differences of the changes in probing pocket depth between the group - iii and group - iv ( bg - ha composite ) suggesting that soft tissue bonding property of bg might have some role in group - iv ( bg - ha composite ) sites . the inter - group comparison between mean gain in ral in the ha , bg , and control sites were found [ table 2 ] to be statistically insignificant suggesting that all three treatment modalities were effective in the treatment of infra bony periodontal defects with a greater trend toward improvement in the bg sites , as also observed by zamet et al . but statistically significant gain ( p<0.05 ) in ral in the group - iv ( porous bg - ha composite ) as compared to group - i sites ( control ) suggesting that strong chemical bonding of hydroxy carbonate apatite layer ( hca ) to collagen created the firm attachment to the host tissue . however , the gain in attachment could be due to new attachment , healing by long junctional epithelium or change in soft tissues post - operatively . although surgical entry is considered to be the most accurate method to assess depth of the defect but it was evaluated through bone sounding because , trauma to regenerated tissue , unavoidable bone loss with each exposure , patient 's discomfort , ethical concerns for re - entry procedure were the limitations of open bone measurements . in the present study , no difference was observed with the bone level measurement recorded through bone sounding and via surgical entry during surgery ( at baseline ) indicating that bone sounding alone was adequate to assess bone defect fill causing minimal trauma to the regenerating tissue . the mean defect fill in the four groups at the end of six months [ table 1 ] falls within the same range or slightly more of the study of laurell et al . dod demonstrated greater defect fill with group - iii and group - iv as compared to the group - ii ( ha ) and group - i ( control ) , but inter group comparison between the group - iii and group - iv showed no statistically significant differences [ table 2 ] in defect fill . it suggested that osteostimulatory property of bg might have some role in group - iv and in improving defect fill noted in this study . in group - i ( control ) sites [ figure 2 , b1 ] , relatively slow radiographic bone fill was seen in iopa radiograph which might be due to early recolonization of cells from the gingival soft tissues to the defect sites and resulted in filling of defects with radiolucent soft connective tissue . in group - ii ( ha ) sites [ figure 2 , b2 ] , apparently slow bone formation which might be due to partial resorption followed by encapsulation of ha particles with radiolucent soft connective tissue . it was also suggested that ha did not appear to stimulate rapid bone growth in the defect but acted simply as an osteoconductive agent . there is no silicon contained in the ha , consequently an incubation period for the ha implant is necessary to accumulate biological hydroxyapatite on the implant , which would delay the deposition process of biological hydroxyapatite on the surface of the implant . in group - iii ( bg , [ figure 2 , b3 ] ) and group - iv ( bg - ha , [ figure 2 , b4 ] ) sites , greater radiographic bone fill in iopa radiograph might be due to osteostimulatory property of bioactive glass . a greater amount of new bone growth was also observed in the bone defects implanted with bioactive glass when compared to group - i ( [ figure 2 ] , b1 -control ) and group - ii ( [ figure 2 ] , b2 -ha ) suggesting that silica rich layer provide multiple nucleation sites for hydroxy - carbonate - apatite precipitation and crystallization . , they produce several growth factors ( igf - ii , tgf- ) and cytokines that modulate osteoblast cell cycle . it resulted in increased osteoblast cell proliferation and differentiation and enhanced synthesis and release of type-1 collagen , alkaline phosphatase and extra cellular matrix ( osteocalcin ) from these cells ; thus , allows rapid bone formation . it is known that mineralization of bone involves a concentration enhancement of silicon at the mineralization front . biologically active sio2 and apatite species was also responsible for inhibiting the proliferation of fibroblasts at bioactive implant interface . an added advantage of bg - ha composite bone graft materials was assumed that ha particles with in the composite bone graft materials acted as a scaffold , encircling which osteoid could deposited that resulted in early increase in strength of newly forming bone . in view of the present finding , both ofd and ofd plus synthetic bone graft particles are effective in the reconstruction of infra bony periodontal defects , but considering the hard tissue measurements it is concluded that bg and bg - ha composite synthetic bone graft particles lead to better results compared to other groups ( group - i and group - ii ) . equally important was the fact that the newly developed indigenous bg and bg - ha composite particles did not cause any extra biological complications and therefore may safely be used as good alternative bone graft materials in humans .
background : several synthetic alloplastic materials have been used in the past as an implant in infrabony defects with a goal to reconstruct the lost part of attachment apparatus via new osseous tissue formation . the present study was undertaken to evaluate and compare clinico - radiographically , the effect of bioactive glass ( bg ) , hydroxyapatite ( ha ) , and bg - ha composite bone graft particles in the treatment of human infra - bony periodontal defects.materials and methods : indigenous synthetic ha , bg , and bg - ha composite bone graft materials were developed in the laboratory . twenty eight infrabony periodontal defects were equally distributed ( i.e. , seven defects ) into four groups . the defects were treated separately with three types of graft materials and non - grafted manner ( open flap debridement alone , control ) to evaluate both the soft and hard tissue responses after six months of surgery . evaluation was done by studying different parameters such as plaque index , gingival index , relative attachment level , probing pocket depth , and radiographic bone fill in intra oral peri - apical radiograph.results:the healing of defects was uneventful and free of any biological complications . the gain in relative attachment level , reduction of probing pocket depth , and bone fill was statistically significant in all four groups . bg and bg - ha synthetic bone graft implanted sites showed significant bone fill ( p<0.05 ) than hydroxyapatite and unimplanted control sites.conclusion:the performance of bg and its composite was better compared to ha and open flap debridement alone for the reconstruction of infrabony defects . the bg - ha composite particles may effectively be used as an alternative bone graft material for infrabony defects .
You are an expert at summarizing long articles. Proceed to summarize the following text: osteoarthritis ( oa ) is a chronic and degenerative joint disease and is considered to be one of the most common musculoskeletal disorders . knee oa is a disease of the elderly that leads to pain and disability , to degeneration of the knee joint , and to worsening of quality of life ( qol ) . the main clinical symptoms are pain , stiffness , edema , deformity , and loss of range of motion , muscle weakness may develop , and even activities of daily life may be hindered by pain as the disease progresses . at the advanced stage , total knee arthroplasty ( tka ) is a common , reliable surgical operation performed at the late stages of oa on patients that are unresponsive to conservative treatment . tka relieves pain and restores function by increasing mobility and correcting any deformity , which consequently helps to improve the qol of the patient both psychologically and physically7,8,9,10,11,12,13 . physiotherapy techniques are the most important tools that patients and doctors have for rapidly mproving qol . however , although the provision of physiotherapy - based rehabilitation after tka is seemingly ubiquitous , its role in restoring mobility and function after surgery remains unclear14,15,16,17 . therefore , evaluation of the effects of physiotherapy programs on qol in patients can provide important information for doctors , physiotherapists , and patients with oa regarding selection of the most appropriate treatment based on patient preferences and convenience . the aim of this study was to investigate the differences in qol of females treated with supervised physiotherapy ( sp ) and a standardized home program ( shp ) after tka for primary oa of the knee . from january 2012 to may 2015 a total of 40 females , who underwent unilateral tka for primary oa of the knee were examined . all the females were advised to undergo tka by a surgeon and met the inclusion criteria , including advanced changes of oa , unrelenting pain , limitation of movement , and muscle contractures , which are unresponsive to conservative treatment . none of the females met the exclusion criteria ( presence of a malignancy , charcot arthropathy , joint infection , advanced osteoporosis , obesity , varices , local infection , or progressing neurological defects ; failure of an abductor muscles procedure ; or n the need for extensive urological or dental procedures . the baseline characteristics of the females are shown in table 1table 1.baseline characteristicssupervised physiotherapystandardized home program patients ( n)2020 age ( yrs)67.9 3.5 ( 59.073.0)*68.2 3.7 ( 59.073.0)*level of education ( n ) : primary school graduates1013secondary school graduates23 university graduates84economic status active / retired ( n ) 4/16 3/17marital status single / married ( n)7/135/15 oa duration ( yr)12.4 2.9 ( 9.019.0)*12.9 2.6 ( 9.019.0)*treatment knee right / left ( n)12/814/6 patients with associated diseases no ( n):20/018/2diabetes35hypertension 43back pain64 osteopenia45depression 31*data are expressed as the mean sd ( minmax ) .. * data are expressed as the mean sd ( minmax ) . the unilateral tka was performed on all the patients by the same surgeon . following combined epidural and spinal anesthesia , the knee was concomitantly prepared and draped with the patient in the supine position . the skin was incised at the midline , and the joint was accessed through a paramedian incision . both femoral and tibial the hospital physiotherapy program included passive knee flexion and extension using a continuous passive motion ( cpm ) machine , which was started on the 1st postoperative day . the rehabilitation program on the first day included ankle dorsiflexion and plantar flexion , isometric exercises for the quadriceps muscle , straight leg raising , active flexion and extension of the knee as tolerated , and isometric and active exercises for hip muscles . on the 2nd postoperative day , the patients sat at the side of a bed and performed active knee flexion and extension exercises , learned how to sit and rise from the bed , and were mobilized to walk with a walker . stair climbing was performed on the 5th day , and the patients were discharged on the 6th postoperative day . after discharge , the patients who underwent tka were randomized on 1:1 ratio to two groups ( sp , n=20 ) and ( shp , n=20 ) using a random number table . the patients in the sp and shp groups participated in a total of 20 sessions of their respective physiotherapy programs for an hour a day , 5 days a week , for 4 weeks ( table 2table 2.components of the supervised physiotherapy and standardized home programsmodalitiessupervised physiotherapy warm - up and stretching ( 10 min ) : 1 . unilateral knee flexion to 90 while standing.5 . climbing on a platform or a flight of stairs.6 . walking backward , on a slope and/or laterally , while crossing the lower limbs.7 . walking in place , with a large amplitude of hip and knee flexion and upper - limb movements.endurance ( 10 min):1 . prone knee flexion.2 . stationary bicycle for range of motion stimulus and endurance.3 . hamstring , quadriceps , and gastrocnemius /soleus stretching.strengthening ( 10 min):1 . straight leg raise.2 . supine submaximal leg press or equivalent ( emphasis on pain - free motion and neuromuscular control vs. pure strengthening).3 . resistive exercises against thera - band while sitting ( starting with 9030 and progressing to 900).balance / gait ( 20 min):1 . braiding ( alternate front and back crossover steps whilst moving laterally ) , progressing by increasing speed.2 . tandem walk forward and backwards.3 . walking with multiple changes in direction on command.4 . all the participants were evaluated by a different physiotherapist , that is , not the one applying the therapy program . qol was examined in all patients with the short form-36 questionnaire ( sf-36 ) in slovakian , and the severity of painwas examined using the scale ( vas ) ; both instruments were administered after daily activities before tka , immediately after physiotherapy ( iap ) , 3 months after tka , and 6 months after tka . the sf-36 questionnaire is divided into 2summary measures , the physical component and the mental component . it includes 36 questions in total , that cover 8 domains : 10 questions for physical function , 2 questions for social behavior , 4 questions for physical role , 2 questions for bodily pain , 5questions for mental health , 3 questions for emotional role , 4 questions for vitality , and 6 questions for general health . the score ranges from 0 to 100 , with the best quality of life corresponding to 100 points and the worst to 0 points . the vas is performed with a 10 cm long line , with the leftmost side representing 0 ( no pain ) and the rightmost side representing 10 ( unbearable pain ) . the participants marked their current level of pain on the scale after normal daily activities . this study was designed in accordance with the rules for human experimental studies and approved by the bioethical committee of the catholic university in ruzomberk . this study also conformed to the principles of the declaration of helsinki ( 1975 , revised 1983 ) . ibm spss statistics version 20.0 ( ibm corp . , armonk , ny , usa ) was used for the statistical analyses . the data are presented as the mean standard deviation ( sd ) , mean difference ( md ) , and 95% confidence interval ( 95% ci ) . two - way repeated - measure analysis of variance ( anova ) was used for comparisons between the sp and shp groups before tka , iap , 3 months after tka , and 6 months after tka and to assess the differences in the variables . the tukey s procedure was used as a post hoc test to identify specific differences between groups at each assessment point . we found the statistically significant improvements in the sf-36 and vas results iap and 3 and 6 months after the tka in both treatment groups ( p<0.05 ) in all patients in both treatment groups ( table 3table 3.intragroup differences from the time before tka until 6 months after tkadomainsupervised physiotherapystandardized home programbefore tkaiap3 mo6 mobefore tkaiap3 mo6 momean ( sd)mean ( sd)mean ( sd)mean ( sd)mean ( sd)mean ( sd)mean ( sd)mean ( sd ) pf15.1 ( 10.6)53.1 ( 10.2)73.9 ( 8.0)87.8 ( 9.9)*14.7 ( 10.0)44.9 ( 8.9)65.3 ( 8.8)80.7 ( 10.6 ) * pr15.7 ( 7.9)51.4 ( 9.7)73.3 ( 7.9 ) 85.3 ( 9.6)*16.2 ( 7.8)44.6 ( 9.9 ) 66.0 ( 8.0)75.7 ( 10.3 ) * gh27.4 ( 9.9)52.1 ( 10.1)71.6 ( 8.2 ) 82.1 ( 9.3)*28.3 ( 9.5)44.3 ( 9.8)65.3 ( 8.5)74.2 ( 7.3)*v29.0 ( 9.5)61.2 ( 7.7)71.5 ( 6.5)81.1 ( 8.9)*29.3 ( 9.3)55.6 ( 9.0)62.3 ( 11.8)75.3 ( 9.3 ) * sf31.2 ( 10.2)62.6 ( 9.4)80.2 ( 8.5)87.8 ( 9.6)*32.3 ( 10.3)55.9 ( 10.7)74.2 ( 9.7)80.4 ( 10.4)*er28.1 ( 11.0 ) 66.9 ( 10.0)82.5 ( 8.0)89.0 ( 7.1)*27.7 ( 10.9)58.6 ( 9.8)74.7 ( 12.1)81.7 ( 7.8 ) * mh46.0 ( 11.4 ) 67.6 ( 10.5)73.4 ( 9.1)81.1 ( 7.9)*45.7 ( 11.3)60.4 ( 10.1)66.1 ( 9.4)74.3 ( 8.7 ) * bp14.7 ( 6.9)53.7 ( 9.3)68.7 ( 7.3 ) 83.1 ( 9.2)*14.1 ( 6.7)46.3 ( 9.7)56.5 ( 8.2)76.1 ( 7.5)*vas7.9 ( 0.8)4.4 ( 1.3)3.0 ( 0.8)1.8 ( 1.3)*7.5 ( 0.9)5.6 ( 1.1)4.3 ( 0.7)2.6 ( 0.9)**statistically significant within group ( p<0.05).before tka : before total knee arthroplasty ; iap : immediately after physiotherapy ; 3 mo : 3 months after tka ; 6 mo : 6 months after tka ; pf : physical function ; pr : physical role ; gh : general health ; v : vitality ; sf : social function ; er : emotional role ; mh : mental health ; bp : bodily pain ; vas : visual analog scale of pain ) . the results post hoc testing with tukey s procedure revealed that there were no significant differences in domains of qol , except for physical role ( pr ) , vitality ( v ) , bodily pain ( bp ) . a statistically significant difference was detected in the pr domain 6 months after tka ( p=0.019 ) , in the v domain 3 months after tka ( p=0.037 ) and in the bp domain 3 months after tka ( p=0.000 ) in favor of the sp group . when the intensity of pain was compared using the vas , a statistically significant difference was detected iap ( p=0.004 ) and 3 months after tka ( p=0.001 ) , and the difference was also in favor of the sp group ( table 4table 4.intergroup differences at each assessment timedomainsupervised physiotherapy vs. standardized home program before tkaiap3 mo6 momd ( 95% ci)md ( 95% ci)md ( 95% ci ) md ( 95% ci ) pf0.4 ( 9.0 ; 9.8)8.2 ( 1.1 ; 17.6)8.6 ( 0.7 ; 18.0 ) 7.1 ( 2.3 ; 16.5)pr 0.5 ( 9.2 ; 8.2 ) 6.8 ( 1.9 ; 15.5)7.3 ( 1.4 ; 16.0)9.6 ( 0.9 ; 18.3)*gh 0.9 ( 9.8 ; 8.0)7.8 ( 1.0 ; 16.7 ) 6.3 ( 2.6 ; 15.2 ) 7.9 ( 1.0 ; 16.8)v0.3 ( 9.1 ; 8.5 ) 5.6 ( 3.2 ; 14.5 ) 9.1 ( 0.3 ; 17.9)*5.8 ( 2.9 ; 14.7)sf1.1 ( 10.7 ; 8.5 ) 6.7 ( 2.8 ; 16.3)6.0 ( 3.6 ; 15.6)7.4 ( 2.2 ; 17.0)er 0.4 ( 9.1 ; 9.9)8.3 ( 1.1 ; 17.8)7.8 ( 1.6 ; 17.3 ) 7.2 ( 2.2 ; 16.7)mh0.3 ( 9.3 ; 9.9 ) 7.2 ( 2.3 ; 16.8 ) 7.3 ( 1.1 ; 15.4 ) 6.8 ( 2.7 ; 16.4)bp 0.6 ( 7.3 ; 8.6)7.4 ( 0.5 ; 15.4 ) 12.2 ( 4.2 ; 20.1 ) * 7.0 ( 0.9 ; 14.9 ) vas 0.3 ( 0.6 ; 1.3 ) 1.2 ( 2.2 ; 0.3)*1.3 ( 2.3 ; 0.4 ) * 0.8 ( 1.8 ; 0.2 ) * statistically significant between groups ( p<0.05).md : mean difference ; 95% ci : 95% confidence interval ; before tka : before total knee arthroplasty ; iap : immediately after physiotherapy ; 3 mo : 3 months after tka ; 6 mo : 6 months after tka ; pf : physical function ; pr : physical role ; gh : general health ; v : vitality ; sf : social function ; er : emotional role ; mh : mental health ; bp : bodily pain ; vas : visual analog scale of pain ) . * statistically significant within group ( p<0.05 ) . before tka : before total knee arthroplasty ; iap : immediately after physiotherapy ; 3 mo : 3 months after tka ; 6 mo : 6 months after tka ; pf : physical function ; pr : physical role ; gh : general health ; v : vitality ; sf : social function ; er : emotional role ; mh : mental health ; bp : bodily pain ; vas : visual analog scale of pain * statistically significant between groups ( p<0.05 ) . md : mean difference ; 95% ci : 95% confidence interval ; before tka : before total knee arthroplasty ; iap : immediately after physiotherapy ; 3 mo : 3 months after tka ; 6 mo : 6 months after tka ; pf : physical function ; pr : physical role ; gh : general health ; v : vitality ; sf : social function ; er : emotional role ; mh : mental health ; bp : bodily pain ; vas : visual analog scale of pain physiotherapy after tka is usually recommended to help patients become functionally independent following knee surgery and to help them return to their pre - disease conditions . the available studies indicate that there is no significant difference between patients treated with sp and those treated with a shp program with respect to functional status of the patient , and overall health status of the patient22,23,24,25 . however , moffet et al.21 found that an intensive sp program between the 2nd and the 4th months after tka was more effective than an shp program in improving short - term and mid - term functional ability after tka . therefore , more intensive rehabilitation should be promoted in the subacute recovery period after tka to optimize functional outcomes in the first year after surgery . furthermore , shields et al.26 found that subjects who received an intensive sp program had fewer problems with work or other daily activities because of physical health ( pr domain ) , had less physical limitation , had fewer disabilities and fewer decrements in well - being , and had higher energy levels and better health than subjects in an shp group . in our study , all the patients showed the significant improvement in the sf-36 and vas results from the time before tka until 6 months after tka . however , there were no significant differences in domains of qol between the sp and shp groups , except for the pr domain 6 months after tka , and the v and bp domains 3 months after tka in favor of the sp group . in our study , we assessed pain with the vas because the sf-36 questionnaire evaluates pain and its effects on qol broadly with a few questions but does not take into consideration intensity of pain . taking the vas results into consideration , the level of pain was consistently reduced from iap to 3 months after tka and persisted until 6 months after tka in the sp group compared with the shp group , and this significantly improved the qol of females who underwent unilateral tka . the qol improvement of our patients from the time before tka until 6 months after tka , may be due to meticulous preoperative interviews conducted prior to surgery and active participation of the patients in the sp or shp program , which led to earlier restoration of mental and physical functions . one of the limitations of the study is inclusion of only female patients who underwent unilateral tka . moreover , our conclusions are limited by the fact that the sample sizes for the sp and shp groups were smaller than would be ideal . therefore , further studies should be carried out on a larger representative sample to verify our results . another limitation of the study is the lack of a long - term comparative analysis of the impact of sp and shp programs on qol . such a study is needed , as it would enable physiotherapists to use the optimal model of therapy in order to obtain the maximum health benefits in people who undergo tka for advanced oa . in conclusion , despite the limitations of the study , their results provide further evidence indicating that patients who undergo tka for primary oa of the knee can achieve a significant improvement in qol by using sp or shp procedures . moreover , the present study provides important information for doctors , physiotherapists , and patients with oa regarding selection of the most appropriate treatment based on patients preferences and convenience .
[ purpose ] osteoarthritis is a chronic and degenerative joint disease and is considered to be one of the most common musculoskeletal disorders . this study evaluated the differences in the quality of life of females treated with supervised physiotherapy and a standardized home program after unilateral total knee arthroplasty . [ subjects and methods ] from january 2012 to may 2015 , a total of 40 females were examined at the central military hospital in ruzomberk , slovakia . quality of life was assessed with the short form-36 . quality of life and intensity of pain after normal daily activity , according to the visual analog scale , were assessed before total knee arthroplasty , immediately after physiotherapy , 3 months after total knee arthroplasty , and 6 months after total knee arthroplasty . [ results ] we found statistically significant improvement of the quality of life results and a decreased intensity of pain at each time point compared with before total knee arthroplasty . [ conclusions ] the results of this study provide further evidence indicating that patients who undergo total knee arthroplasty for primary osteoarthritis of the knee can achieve a significant improvement in the quality of life by using supervised physiotherapy compared with a standardized home program .
You are an expert at summarizing long articles. Proceed to summarize the following text: dexmedetomidine ( dex ) is a highly selective 2 receptor agonist with sedation , analgesia , and anxiolytic in clinical practice [ 1 , 2 ] . it has an analgesic - sparing effect , significantly reducing opioid requirements in both intraoperative and postoperative period . dex also has a sympatholytic effect , which can reduce the stress response to surgery and ensure a stable hemodynamic state . few of clinical studies have explored the use of intravenous dex in attenuation of stress and inflammatory response and postoperative pain . in a pediatric cardiac surgery study , dex at a 1 g / kg loading dose followed by infusion at 0.5 g / kg / h until separation from cardiopulmonary bypass , which was effective in blunting stress and inflammatory response to surgical trauma as indicated by lower norepinephrine level , il-6 , and il-10 ratio , while achieving better postoperative pain control . in another pediatric cardiac surgery study using caudal bupivacaine 0.25% , 2.5 mg / kg and dex 0.5 g / kg attenuated stress response to surgical trauma and provided better postoperative analgesia . in view of these consistent clinical effects , intranasal dex is convenient , effective , and noninvasive and also has useful analgesic and sedative effects in surgical procedures . cheung 's research has shown that intranasal dex 1 and 1.5 g / kg in surgical procedures produced significant sedation and less postoperative pain [ 7 , 8 ] . however , the clinical use of intranasal dex for the stress and inflammatory response relief affection in surgical procedures has not been explored . we therefore conducted this prospective , randomized , and double blind study to explore the efficacy of intranasal dex in regard to operative conditions , the stress and inflammatory response relief , and postoperative analgesia following fess . this prospective , randomized , and double blind clinical trial was approved by the ethics committee of anhui provincial hospital , anhui medical university , and registered at chinese clinical trial registry ( chictr ) with registration number chictr - trc-14004886 . patients of either sex with the american society anesthesiologists physical status i - ii , aged between 18 and 60 years , undergoing fess under la were recruited . exclusion criteria were pregnancy , history of serious adverse reaction , or patients with known sensitivity to local anesthetic drug and allergy to study drugs , patients with bmi > 35 kg / m , ischemic heart disease , asthma , sleep apnoea syndrome , use of 2-agonists or antagonists , hypotension ( baseline systolic arterial pressure ( sap ) , 100 mmhg ) , and bradycardia ( baseline heart rate ( hr ) , 60 beats / min ) , patients on pain perception modifying drugs , and those with history usage of any opioid or sedative medications in the week prior to surgery . the visual analogue scale ( vas ) ( 0 , no discomfort and no pain ; 10 , a high level of discomfort and maximum pain ) was explained to the patients during the preoperative visit . the patients were randomized to the two study groups by random number table method , which was prepared by an unwitting statistician , placebo group , and dex group ( n = 30 ) . all patients were sent to the anesthesia preoperative room without any premedication 60 min before the surgery . standard monitoring consisted of five - lead electrocardiography ( ecg ) , oxygen saturation ( spo2 ) , and noninvasive blood pressure . one anesthesiologist who was unaware of the clinical nature of the study monitored and conducted the case . study drug , either undiluted dex 1.5 g / kg ( dex group ) or the same volume of 0.9% normal saline ( placebo group ) , was administered to each naris as drops . one anesthesia nurse who was not involved in the experiment prepared all the study drugs . the anesthesiologist in the operating room who was blinded to group assignment collected the date . one hour after study drug administration , patients were then transferred to the operating room for the surgical procedure . an infusion of ringer 's solution 8~10 ml / kg was given after a 20-gauge intravenous cannula was inserted in the dorsum of each patient 's left hand . all the operations were done by the same surgical team of three otolaryngology surgeons , who also performed the la with the standard fess surgical technique in both patient study groups before the surgical procedure that involves submucosal infiltrative anesthesia with the 2 ml mixture of 1% lidocaine and epinephrine ( 1 : 100,000 ) in the region of the agger nasi cell and the middle turbinate ; then packed nasal passages and total nasal passages with cotton pieces contain 1% tetracaine 20 ml and 1 adrenaline 8 ml under nasal endoscope . after confirming adequate analgesia , the surgical procedure systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , hr , and spo2 were recorded at seven time points ( t0 , baseline ; t1 , 5 min after intranasal instillation ; t2 , 30 min after intranasal instillation ; t3 , before local anesthesia ; t4 , the beginning of the operation ; t5 , 30 min after operation ; t6 , the end of operation ; t7 , out of pacu ) . after the procedure , patients were transferred to the pacu and hemodynamic parameters ; degree of analgesia was monitored until transferred to surgical ward . patients ' body movements leading a temporary stop to surgical procedure , caused by pain or discomfort during procedure , were also recorded . quality of intraoperative surgical field during fess was evaluated by the surgeons with formmer 's scores of surgical field quality ( 1 , bleeding , so mild it was not even a surgical nuisance ; 2 , moderate bleeding , a nuisance but without interference ; 3 , moderate bleeding that moderately compromised surgical ; 4 , bleeding , heavy but controllable , that significantly interfered ; 5 , massive uncontrollable bleeding ) . the degree of adverse reactions of hemostatic stuffing after fess was also evaluated ( 1 , no swelling , can tolerate ; 2 , swelling , can barely tolerate ; 3 , swelling , can not tolerate ) . surgeons who were asked to evaluate their overall satisfaction with the procedure ( immediately after the surgery ) , and patients who were also asked to evaluate their overall satisfaction with the procedure ( when shifted to surgical ward and 48 h after the procedure ) by numerical rating scale ( nrs ; 0 , being least satisfied ; 10 , being most satisfied ) . the standard vas score was used to evaluate postoperative pain at 2 h , 4 h , 8 h , 12 h , 24 h , and 48 h postoperatively . three milliliter of venous blood was collected on the morning of the operation day and arrival in the pacu . the rest was added to tubes without anticoagulant , perfectly still until the serum separation , the serum precipitated was taken with centrifugal to centrifuge at 4000 rpm in 4c for 10 min , and then the supernatant was sucked out to place in 80c cryogenic refrigerator to wait to test epinephrine , norepinephrine , interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , and tumor necrosis factor - alpha ( tnf- ) . epinephrine and norepinephrine were assayed by enzyme - linked immunosorbent assay ( elisa ) ( 2-cat ; elabscience biotechnology co. , ltd ) . il-6 , il-8 , and tnf- was measured with the immulite automated chemiluminometer ( siemens healthcare diagnostics , deerfield , il ) . our primary outcome measures were stress hormones and inflammatory markers levels ; the secondary outcomes were postoperative amplification of pain sensation and discomfort with hemostatic stuffing . the sample size estimation was based according to norepinephrine , the most intuitionistic one of the stress hormones . a pilot study of 10 patients at our center found that the mean standard deviation ( sd ) of norepinephrine for fess was 466.94 73.74 . a reduction of 10% in norepinephrine ( 420.25 66.37 ) after intranasal dex in the treatment group was considered clinically significant , and this required a sample size of 29 per group to achieve a power of 80% and a type i error of 5% . to compensate for the possibility of dropout , we recruited 60 patients , 30 patients per group . the vas pain scores over the 48 postoperative hours are expressed as auc using the trapezoid rule and were analyzed by the mann - whitney u test . the demographic characteristics data were evaluated using unpaired t - test for between - group and paired t - test for within group comparisons . data are presented as mean sd ( sem ) , count ( % ) , or as median ( iqr ( range ) ) . no significant difference was seen in demographic data , surgical characteristics , and duration of operation between the two study groups . however , intranasal dex resulted in a significant reduction in blood loss ( p = 0.030 ) . plasma concentrations of epinephrine , norepinephrine , and blood glucose before operation and after operation are displayed in figure 2 . all were with no difference between the groups before operation and they all increased in both groups after operation compared to before operation , but the difference of the three variables was significant in placebo group in contrast with only the blood glucose in dex group ( p < 0.0001 ) . between - group comparison of epinephrine , norepinephrine , and blood glucose after operation and dex group was lower than that of placebo group ( at p = 0.0063 , p < 0.0001 , and p < 0.0001 , resp . ) . plasma concentrations of il-6 , il-8 , and tnf- were with no difference between the groups during preoperation and they all increased in both groups after operation compared to before operation , and the difference of il-6 and tnf- was significant in contrast with no difference about il-8 in both groups . il-6 and tnf- were significantly lower in dex group after operation ( at p < 0.0001 and p = 0.0013 , resp . ) ( figure 3 ) . more patients from dex group than placebo group could tolerate the hemostatic stuffing ( p = 0.031 ) . mean auc of vas pain scores for postoperative 212 h were 26.2 and 39.4 for patients in groups dex and placebo , respectively ( p < 0.001 ) ; while mean auc of vas pain scores for postoperative 1248 h were 139.2 and 145.4 for patients in groups dex and placebo , respectively ( p = 0.741 ) ( figure 4 ) . although the auc of postoperative vas pain scores at 212 h was significantly lower in dex group , no similar pain relief was noticed for postoperative auc vas at 1248 h. there was no difference about sap , dbp , hr , and rate pressure product ( rpp ) at baseline between the two groups , and they both had a rise over time during the operation ( from t3 to t6 ) . however , the difference about hr and rpp was significant in placebo group ( p < 0.0001 ) . between - group comparison of hemodynamic variables at the same time period after intranasal drugs ( from t3 to t7 ) and dex group was lower compared to placebo group ( at p < 0.05 and p < 0.0001 , resp . ) ( figure 5 ) . concerning the surgical field quality , more surgeons from dex group than placebo group were satisfied ( p = 0.046 ) ( table 3 ) . due to pain or discomfort , more patients from placebo group than dex group had body movements that affected the normal operation ( p = 0.035 ) ( table 4 ) . when asked about satisfaction with the procedure , both surgeons and patients from dex group felt better and would use the same drug again ( at p < 0.001 and p < 0.001 , resp . ) ( table 5 ) . this randomized , double - blinded , and comparative study was undertaken to evaluate the use of intranasal dex in fess . our principal findings were that patients given intranasal dex during fess were safe and effective and experienced significantly slighter hemodynamic changes and perioperative stress and inflammatory response , less postoperative discomfort , and pain in the early postoperative period , and the surgeons also had better surgical field and more satisfaction with its use . fess is usually performed under local anesthesia , which is always interrupted because of the fear , anxiety , pain , discomfort , and cardiovascular stress of patients , and the complications such as bleeding and postoperative pain are always existent . the modification of stress and inflammatory response may potentially be useful in attenuating the postoperative pain and discomfort and improve the postoperative outcome . recently , monitored anesthesia care ( mac ) and general anesthesia ( ga ) are rapidly used but they both have their defects such as the risk of respiratory depression and the high medical costs [ 14 , 15 ] . considering the hemorrhagic secretions into the mouth during the process of the surgery , many surgeons do not want to give patients additional anesthesia sedatives ; they need the patients ' cooperation to spit the hemorrhagic secretions . anyway , fess could be done under la without any sedatives safely and feasibly , despite more or less a bit discomfort [ 16 , 17 ] . intravenous dex in ent surgeries like functional endoscopic sinus surgery ( fess ) , middle ear surgeries , thyroplasty , and septoplasty under monitored anesthesia care ( mac ) has been reported to be safe and effective [ 1821 ] . although dex has both sedative and analgesic properties and has been used as a single agent in many painful procedures , the analgesic potential , however , does not approximate the potency of opioids , many patients who received dex required supplemental analgesia . however , using dex in a multimodal manner may be useful to allow lower doses of each component drug and , potentially , side effects similar to the way it is used during general anesthesia . changes in hemodynamic properties in response to surgical stress , such as increase in blood pressure , hr , and rpp were significantly lower in dex group . these decreases may be useful for clinic that significantly reduced the need for vasopressors or anticholinergic support . our results are similar to other studies which lower hr and blood pressure that were observed in the dex group [ 7 , 8 ] . we tested the plasma stress hormones to verify the theory and found that plasma epinephrine , norepinephrine , and blood glucose were indeed significantly lower in dex group . fess is a commonly performed procedure ; however , close proximity of the surgical field to major blood vessels makes the surgical field associated with blood loss easy . the stable and lower hemodynamics made the surgical field associated with less blood loss in dex group . in view of this , surgeons could do the operation better and faster , and most of surgeons from the dex group were satisfied with its use . these findings suggest that the combined effect of intranasal dex and local anesthesia in reducing the response to surgical stress and inducing hypotension may minimize the risk of surgical complications and stabilize hemodynamics for fess . clinical studies have shown that the acute pain caused by fess could lead patients to sleep deprivation [ 15 , 26 ] . also postoperative pain has become one of the most important factors that affect patients ' postoperative recovery . postoperative pain due to fess should be managed aggressively as the earlier treatment of analgesia , the better the results [ 27 , 28 ] . previous studies have suggested that postoperative pain of nasal endoscope is mainly due to the stimulation of the surgery itself . surgical trauma leads to the release of inflammatory mediators from immune cells and nonneuronal cells in the periphery , directly causes pain . the proinflammatory cytokines ( pics ) such as interleukin-6 ( il-6 ) and tumor necrosis factor- ( tnf- ) are an important group of inflammatory mediators and play an essential role in pain sensitization [ 29 , 30 ] . the peripheral effects of these pics on sensitizing nociceptors have been well documented . on the one hand , part of which is passed to the nerve center of the more advanced , produced reaction on the spinal cord and cortical , which eventually form the pain experience ; on the other hand , part of the peripheral nociceptive stimulus can directly lead to central sensitization and super reactivity to cause more severe pain . therefore , systemic or regional analgesic regimens initiated before the onset of surgery can prevent both peripheral and central sensitization , thereby attenuating the postoperative amplification of pain sensation [ 27 , 31 , 32 ] . significantly increased il-6 and tnf- serum levels have been detected in our patients after operation in both groups . this result is consistent with our findings of postoperative vas pain scores , which dex group also had a significantly lower vas pain scores for postoperative 212 h. therefore , we think that our results could give a suggestion that the beneficial effects of intranasal dex initiated before the onset of surgery would be effective to reduce the inflammatory response , so as to reduce the postoperative pain sensitization . as the experience of the fess of the author himself , the postoperative pain mainly occurs during the postoperative 24 h , the nasal cavity - filling period . nasal cavity filling is an important means of fess postoperative hemostasis ; however , nasal cavity filling may inevitably have some adverse reactions , such as elevated intraocular pressure , and difficulty in swallowing . furthermore , long time mouth breathing could cause postoperative sore throat and influence the patient 's sleep . then the patients ' mood will be worse and lead to the pain sensitization aggravating . our research suggests that intranasal dex initiated before the onset of fess could significantly reduce the postoperative discomfort . not only the surgeons but also the patients in dex group were generally satisfied with the procedure . more optimal operating conditions , less blood loss , and better cooperation of patients got the surgeons ' approval ; moderate sedation , slight adverse reaction of hemostatic stuffing , and less postoperative pain also improved the patients ' satisfaction . in conclusion , intranasal dex with local anesthesia used in fess could yield more stable hemodynamic profile and lower stress and inflammatory response and attenuate the adverse reactions of hemostatic stuffing and pain sensitization at the early postoperative period , leading to better surgical field and satisfaction with surgeons and patients . stress - free anesthesia , with measurement - based control of surgery stress , would improve postoperative outcome .
background . a strong ongoing intraoperative stress response can cause serious adverse reactions and affect the postoperative outcome . this study evaluated the effect of intranasally administered dexmedetomidine ( dex ) in combination with local anesthesia ( la ) on the relief of stress and the inflammatory response during functional endoscopic sinus surgery ( fess ) . methods . sixty patients undergoing fess were randomly allocated to receive either intranasal dex ( dex group ) or intranasal saline ( placebo group ) 1 h before surgery . stress hormones , inflammatory markers , postoperative pain relief , hemodynamic variables , blood loss , surgical field quality , body movements , and satisfaction were assessed . results . plasma epinephrine , norepinephrine , and blood glucose levels were significantly lower in dex group as were the plasma il-6 and tnf- levels ( p < 0.05 ) . the weighted areas under the curve ( aucw ) of the vas scores were also significantly lower in dex group at 212 h after surgery ( p < 0.001 ) . furthermore , hemodynamic variables , blood loss , body movements , discomfort with hemostatic stuffing , surgical field quality , and satisfaction scores of patients and surgeons were significantly better ( p < 0.05 ) in dex group . conclusions . patients receiving intranasal dex with la for fess exhibited less perioperative stress and inflammatory response as well as better postoperative comfort with hemostatic stuffing and analgesia .
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Proceed to summarize the following text: as characterized by hanchanale and coworkers , the perception of botulinum toxin a has been transformed from poison to a healing agent . information concerning botulinum neurotoxin serotypes , molecular structures , substrate specificities , mechanisms of zinc - dependent peptide hydrolysis , ion channel formation , and other detailed topics has been extensively reviewed [ 24 ] . of the seven immunologically distinct serotypes ( a f ) from clostridium botulinum and several other species , type a is the best characterized and is among the most potent of all toxins . the neurotoxin is initially expressed as a single polypeptide of nearly 1300 amino acid residues ( mw ~150 kda ) . crude toxin extracts ( mw ranges from ~300 to 900 kda ) contain several nontoxic ancillary proteins that form a complex with the neurotoxin . when ingested , these additional proteins are thought to protect the neurotoxin against austere environments such as those found in the certain regions of the gastrointestinal tract . the neurotoxin is posttranslationally modified to form two chains that are covalently bridged with a disulfide bond . the light ( l ) chain ( mw ~50 kda ) has zinc - dependent proteolytic activity , while the heavy ( h ) chain contains the translocation and binding domains . subsequent to binding to specific receptors ( sv2 ) at peripheral cholinergic nerve terminals , the receptor - toxin complex is internalized into a membrane - bound compartment that undergoes a drop in ph . conformational changes occur that allow the insertion of the h - chain into this compartment 's membrane . as a result , the disulfide bond that links the l and h chains is reduced , an ion channel is formed , and the presumed proteolytic active moiety , the l - chain , is translocated into the neuroplasm . when the type a toxin substrate , snap-25 , is selectively cleaved , synaptic vesicle - mediated neurotransmission is blocked that could eventually lead to fatal paralysis . since the 1980s , the therapeutic potential of this toxin has been exploited . extraocular muscles have been injected with the partially purified neurotoxin as an adjunct or alternative to surgical correction in treating strabismus [ 7 , 8 ] . the chemodenervation effects of this most poisonous of poisons have been used to relax hyperkinetic striated muscle groups to diminish the effects of dystonia and related diseases . food and drug administration ( fda ) for the following indications : strabismus , blepharospasm , cervical dystonia , upper limb spasticity , maxillary hyperhidrosis , chronic migraine , and urinary incontinence . these indications along with the temporary enhancement in the appearance ( cosmesis ) with botox cosmetic of moderate to severe wrinkles in adults [ 13 , 14 ] introduces the theme of muscle immobilization in terms of a desired therapeutic outcome . advantage has also been taken of this toxin 's chemoimmobilization property to improve the healing of wounds . in contrast to the relatively vast amounts of information regarding this toxin 's structure and mechanism of action , the newer , off - label uses for botulinum toxin have been less extensively reviewed . to gain further insight regarding the scope of these efforts , we have gathered and examined biomedical research articles by conducting systematic searches of the relevant literature in pubmed , in a manner similar to that of steele and madoff . we have examined studies that range from descriptive observations to randomized controlled clinical trials to obtain more information about the components and processes involved in wound healing and the related time courses of action of botulinum toxin a. the processes observed clinically on the wound healing effects of the type a toxin are at an early stage of our understanding . this proposal is substantiated by evidence - based reviews that critically evaluate this toxin 's effects with different indications [ 17 , 18 ] . we previously noted [ 19 , 20 ] that only a few clinical studies have focused on kinetic analyses . constructing even partial models for the clinically observed effects by this toxin remains a challenge . to advance our understanding , we have selected some of those clinical studies that have examined the timing of this toxin 's effects . botxminer , the botulinum reference tool of clostridial neurotoxin citations in entrez - pubmed / medline , was initially used to search in article titles , abstracts , and mesh headings for the words wound and heal or healing . a more extensive list of 29 wound - related keywords was then generated : anal , angiogenesis , collagen , cytokine , fibroblast , fibroblastic , fibrosis , fissure , flap , glycosaminoglycan , heal , healing , hemorrhoid , hemorrhoidectomy , hypertrophic , incision , inflammation , inflammatory , keloid , lesion , repair , scar , scarring , sphincterotomy , surgical , tendon , tensile , ulcer , and wound . this controlled vocabulary was used in the batch mode to search botxminer for additional related citations . another set of filter terms were used to find time - course - related information about this toxin . this set included 26 terms : clearance , day , decay , decline , delay , diffusion , duration , follow up , frequency , hour , hr , interval , kinetic , latency , minute , month , onset , period , persistence , recurrence , repeat , resistance , sec , time , week , and year . within the two lists in the batch matrix search , ( a1 , a2 , , am ) and ( b1 , b2 , , bn ) , the terms undergo the or operation ( ) which can be represented in a general form of ( a1 or a2 or , etc . ) . in addition , the lists are combined with the and operation ( ) which results in a batch query that can be represented by ( 1)(a1a2am)(b1b2bn ) . summaries from the two sets of terms were returned by botxminer in the form of tables , histograms , and lists . additional keywords and phrases within the more than 70 downloaded text files were automatically searched with file search assistant ( v. 3.1 , 2009 , aks - labs , raleigh , nc , usa ) . data from the clinical literature that were analyzed were fitted to an exponential function ( 2)y = y0+(a)exp(kdecayt ) , where y is the cumulative number of patients who are free of symptoms at time = t , y0 is the cumulative number of patients who are symptom - free at t = , a is a preexponential constant , and kdecay is the rate constant for the decay of this response . chicago , ill , usa ) was used to conduct a least - squared fit for the values of y0 , a , and kdecay . this equation is commonly used for simulating the decay rate of a reactant from a single model compartment . the fda has approved generic , nonproprietary names for commercial formulations of botulinum toxin [ 2224 ] . for botulinum toxin type a , botox ( allergan , calif , usa ) is onabotulinumtoxina , dysport ( ipsen biopharm limited co. , uk ) is abobotulinumtoxina , and xeomin ( merz pharma gmbh & co kgaa . , germany ) is incobotulinumtoxina . for botulinum toxin type b , myobloc / neurobloc ( solstice neuroscience , inc . , pa , usa ; eisai ltd . , uk ) is now rimabotulinum toxin b . the changes in nomenclature emphasize the different potencies and the noninterchangeable unit dosages of these distinct brand name products . as reviewed by alberto , these distinctions in names emphasize the differences in manufacturing and formulation techniques that may contribute to differences in the pharmacokinetics , efficacy , safety , and antigenicity among these products . in the present paper , partially purified toxin with nontoxic or accessory proteins is referred to as botulinum toxin type a to distinguish it from botulinum neurotoxin a ( bont / a ) , the pure holotoxin . the lists of selective keywords are not intended to be exhaustive but serve as a starting point for the present work . additional terms , such as proctology and coloproctology , can be used in more comprehensive studies . verification confirms that , for example , the equations being coded are producing the correct calculations . software can be validated when it can model the results that best fit existing data , and is subsequently reinforced by further data obtained experimentally . for kinetically related clinical problems , the underlying processes that need to be included are still uncertain . furthermore , mathematical models are not designed to replace validation by basic research experiments or clinical observations . rather , models are meant to enhance validation procedures by providing stimuli for new ideas , hypotheses , and perspectives on the problems being examined . a preliminary search of botxminer , using the query wound and ( heal or healing ) for years 19802010 , returned over 150 citations about half of which were true positives due to the large number of references related to tetanus ( false positives ) . from the list of true positive citations , a variety of indications were found , in which botulinum toxin a has been used for wound healing and related conditions . these examples included experimental cutaneous scars in animal models [ 15 , 28 ] and in clinical studies : chronic anal fissures [ 29 , 30 ] , cleft lip surgical repair , traumatic head lacerations or elective excisions of forehead masses , focal fold granuloma , hypertrophic scarring , pressure ulcers [ 35 , 36 ] , raynaud 's phenomenon ( vasospastic ischemia of the digits , digital ischemia , including chronic ulcers ) , and self - mutilation injuries in lesch - nyhan syndrome . another healing application of botulinum toxin a , referred to as protective ptosis , is used against persistent corneal ulcers , burns , and other ophthalmic - related problems [ 39 , 40 ] . conducting a matrix batch search in which tetanus was filtered out , using 26 time - related terms along with the 29 wound healing - related terms , yielded 671 unique citations . from this filtered output , we concentrated on references dealing with wound conditions and indications in which botulinum has been used for therapeutic purposes in which some mechanistic , dosage , and/or kinetic information was also available ( methods , figure 1 ) . the normal wound - healing process has been described as being comprised of four overlapping phases : haemostasis , inflammation , tissue proliferation , and remodeling . if any of these processes are disrupted , healing is impeded leading to a chronic wound state . the interference by botulinum toxin in reducing muscle movement has helped to define healing phases further . for example , a vicious cycle involving inflammation , pain , and muscle spasm was first noted to be the underlying cause for the development of chronic anal fissures [ 29 , 42 ] . subsequently , low blood flow and ischemia were added to this cycle [ 4345 ] . additional components of the healing process have been identified for other conditions as presented in this section . scar formation is a hallmark of wound healing , and it usually causes significant physical , psychological , and cosmetic problems . hypertrophic scarring is a common , refractory dermal disease that is manifested by the abnormal appearance of wound healing which can be the result of different types of injuries [ 34 , 46 , 47 ] . in the study by xiao et al . , 19 patients were treated once a month over 3 months with 1.835 u botulinum toxin a ( hengli / cbtx - a , lanzou biochemical co. , china ) . scores were assigned before treatment for the associated erythema , pliability ( lesion softening ) , and the severity of itching . after a 6-month followup of the 19 patients participating in that study , 15 gave an overall assessment of their lesion improvement as good , and seven others rated their improvement as excellent . some critical comments provided by the authors included the lack of control subjects , the study was not double blinded , and a small patient population size . finally , there was only a relatively short follow - up time of 6 months so that no determination of the total time course of toxin action could be established . a number of quantitative parameters may be accessed to evaluate the effectiveness of wound healing by botulinum toxin a. increased metabolic activity and inflammation during the healing process induce muscle contractions around the edges of the skin wound [ 32 , 46 ] . the major role of botulinum toxin a in this healing process is to prevent the repeated , small contractions that produce microtraumas near the hypertrophic scar and thereby decrease the tensile force ( muscle activity ) during scar formation . traditional surgical techniques that align incisions along langer 's lines do not prevent repeated contractions . the development of fibrosis also involves the deposition of extracellular collagen and glycosaminoglycans that can cause the scar to hypertrophy , invert , and become hyperpigmented resulting in poor color matching of this tissue with the neighboring skin [ 15 , 28 ] . other parameters for wound healing include size of wound , amount of and infiltration of inflammatory cells , blood vessel proliferation , and wound thickness . additional cellular and molecular mechanisms of healing by the formation of scar tissue ( traumatic cicatrisation ) are beginning to be elucidated . transforming growth factor 1 ( tgf- 1 ) is a fibrotic cytokine that stimulates cellular growth , differentiation , and adherence and leads to the collagen deposition . this cytokine initiates these processes by extracellularly binding to a coupled pair of serine - threonine kinases . on binding , one receptor recruits and phosphorylates the other this signaling pathway eventually stimulates transcription of the collagen gene and the formation of hypertrophic scars . because human fibroblasts derived from hypertrophic scars overexpress and secrete tgf-1 , another wound - healing effect of botulinum toxin a has been speculated to be inhibiting the secretion of tgf-1 . similarly , suramin , an antifibrotic polysulfonated naphthylurea compound , has been reported to promote wound healing by antagonizing tfg-1 in muscle - derived fibroblasts . the circulatory system is also affected by the apparent ability of botulinum toxin to enhance wound healing . using a rat model for wounds , yoo 's group observed that pretreatment with botulinum toxin a increased dorsal skin flap survival and concluded that this process was caused by increased perfusion . because this toxin inhibits secretion of norepinephrine from sympathetic vasodilator and vasoconstrictor neurons [ 51 , 52 ] , the effect of botulinum toxin a may involve increased perfusion by decreasing sympathetic vasoconstriction in the skin flaps , thus promoting skin flap survival . the protective effects of botulinum toxin - induced ptosis have been used for the conditions of recalcitrant corneal ulcers and other surface disorders as an alternative to the surgical practice of partially sewing the eyelids together ( tarsorrhaphy ) . this secondary healing effect is produced when botulinum toxin a is injected into eye muscles , typically the levator palpebrae superioris ( lps ) muscles . this therapeutic application has been the subject of studies that have also generated kinetic data . from an open - label , multicentered study with 16 ophthalmic patients who received 5 u botox in the lps muscle , the time to suitable ptosis was 4.0 0.5 days ( mean se , range : 28 days ) , and the duration of this ptosis was 46.0 12 days ( 1206 days ) . a similar number of patients who received a single , lower dose ( 2.5 u ) had a comparable mean time to ptosis ( 3.6 days ) and a shorter , mean duration ( 16 days ) . although statistical analyses with more patients are required to make more definitive conclusions , these trends are not unexpected but are , nevertheless , remarkable because a twofold change of a low dose apparently resulted in appreciable differences in duration . chronic anal fissure ( caf ) is a painful condition caused by spasms of the internal sphincter smooth muscles . the traditional surgical approach has been sphincterotomy that can result in the adverse effect of incontinence . traditional nonsurgical approaches that have been used include sitz bath , topical anaesthesia , nitroglycerin , isosorbide dinitrate , nifedipine , diltiazem , l - arginine gel , hyperbaric oxygen , and botulinum toxin a . the first use of botulinum toxin as a medical alternative for caf was conducted by jost and schimrigk . for this indication , we chose what has been described as one of the longest follow - up studies using botulinum toxin ( type a ) in the treatment of caf . we analyzed data for the time course of recurrence of caf symptoms in patients in a 42-month follow - up study [ 55 , table 1 ] . as illustrated in figure 3 , at the beginning of the followup ( at t = 0 ) , all 53 patients were symptom - free for 6 months after one to two injections of 1020 u botox . most patients ( 31 of 53 ) did not undergo recurrence indicating that they remained healed during this follow - up period . from the nonlinear fit of these data to ( 2 ) , the fitted values sems are y0 = 29.2 1.2 , a = 24.4 1.3 . the kdecay ( decay rate constant ) is 7.08 10 1.07 10 month , 1.64 10min ( see below ) , or 0.85 years ( see table 1 ) . the value of 1/kdecay or is 14.1 months , and the corresponding value of t1/2 ( = ln2 ) is 9.8 months or 294 days . the long - term success rate was only 31/53 ( 58% ) or a 42% rate of recurrence . from arroyo 's group , recurrence rate of 12% occurred initially , and a rate of 53% at the 3-year follow - up point . bilateral fissurectomies of the internal anal sphincter combined with toxin injections have been reported to have a high success rate although the follow - up time in that study was limited to 1 year . also , it was not determined whether surgery itself produced a similar rate of healing . an interpretation of the single exponential decay curve during the 42-month followup ( figure 3 ) is that it reflects a zero - order elimination or inactivation step of the persistent , intraneuronally located toxin . this step is described by the rate constant of decay , kdecay , in figure 4 and ke in a previous publication . the table summarizes the processes displayed in figure 4 and highlights the recurrence of symptoms that may be used to gauge the slow elimination or inactivation of the type a botulinum toxin . notably , this rate may be comparable to the slow rates of healing of some chronic wounds . alternatively , the long - lived toxic effect may be related to the rate of restoration of intact snap-25 intracellular levels or a combination of a slow degradation and a persistent inhibitory action of the snap-25 bont / a cleavage product . the value of t1/2 of 294 days is comparable to the recurrence times of 444 132 days ( mean s.d . ; range 270718 days ) for achalasia patients after receiving a single injection of 80 u botox . on the other hand , our calculated decay rate constant of 1.64 10min is about 1000 times slower than the estimated rate constant for decay of efficacy ( 1.1 10min ) in a single dystonic patient [ 58 , 61 , 62 ] . this difference may be due to several factors , among which are the different patient populations , the muscles being injected , the conditions being treated , and the assessment methods . while the above studies show encouraging trends in support of using botulinum toxin a in wound - healing paradigms , additional studies are necessary . overall , more prospective clinical studies of these treatments with botulinum toxin a are needed . evidence from blinded , randomized , placebo - controlled , multicentered studies will help determine if these toxin treatments have significant benefit and if the minimal adverse reactions can be sustained . future trials should also use larger populations of more homogeneous ( standardized ) patients and control subjects , plan to examine long - term outcomes , and conduct cost - benefit analyses . although randomized controlled trials are considered the gold standard of clinical research , assessing them using criteria for standardizing phase iii trials remains a substantial challenge . moreover , there is also a need for additional controlled studies to clearly establish an advantage of botulinum products over other methods . an outcome of one of these analyses was the low probability that type a botulinum toxin or calcium channel blockers were found to be more effective in treating caf than nitroglycerin ointment in 182 patients . 's analysis which determined that for 279 caf patients the traditional surgical procedure of lateral internal sphincterotomy ( lis ) was more effective than botox in healing chronic anal fissure . while lis produced a higher rate of minor anal incontinence , botulinum toxin was associated with a higher rate of recurrent disease . for those patients who had a high risk of incontinence , local injection of the toxin computational modeling and simulation studies at different levels of granularity ( i.e. , detail ) should also be beneficial . starting with existing minimal kinetic models [ 19 , 66 ] , dose - dependent kinetic models could be developed to predict the time course ( onset , duration , and recurrence of symptoms ) and the extent of botulinum toxin a 's effectiveness . kinetic models could help to identify what research gaps exist and which ones can be experimentally or clinically resolved . one gap that could be experimentally verified is to determine if the intracellular diffusion of botulinum toxin a is influenced by other coinjected materials , for example , epinephrine and local anesthetics ( lidocaine , xylocaine ) , compounds that have been considered in controlling local diffusion and predicting the extent of this toxin 's paralytic effect [ 15 , 32 ] . as more realistic physiological - pharmacological models are developed , more free parameters and more sources of error , assumptions , and caveats will need to be evaluated . potentially confounding factors associated with wounds include cell viability , alkali conditions , the generation of reactive oxygen species , and inflammation that is related to low blood flow and ischemia . changes in the ion flow through transmembrane channels and in metabolism could also have roles in wound healing . another confounding factor is the well - known , persistent muscle weakness ( up to 5 years ) that results from protracted patient immobilization due to critical illness polyneuropathy or myopathy [ 68 , 69 ] . this persisting weakness and residual neurologic deficits are likely due to denervation , combined with catabolic muscle wasting and potential myopathic changes . it remains for future studies to differentiate these physiological deficits from botulinum toxin - induced effects in either a botulism patient or with therapeutic treatments with this toxin . the tissues involved in the healing process in cytoskeletal architectures and in membrane structures associated with various organs also need to be considered . neurophysiological abnormalities , such as transient denervation - induced muscle fibrillations and presynaptic alterations producing muscle fasciculations , may also need to be considered . other complicating factors in the healing process could also involve the biomechanical dynamic properties of soft tissue ( e.g. , stiffness ) in response to stress and strain , and tissue anisotropy ( directionality of nonhomogeneous material ) . this succinct review examines the soft tissue wound - healing properties of botulinum toxin . when viewed from the perspective of treating neurologic and other disorders , it is noteworthy that the efficacy of this toxin is predicted to be transient as the toxin 's effect wanes , while for wound healing , a permanent resolution is expected . further , with respect to wound healing , the concept is described that for some lesions , botulinum toxin interferes with the vicious cycle of muscle spasm , pain , inflammation , decreased blood flow , and ischemia . a reaction pathway scheme is outlined to illustrate botulinum toxin 's involvement in stopping the vicious cycle . a minimal kinetic scheme for healing chronic wounds is also presented that includes different macroscopic states of soft tissue conditions ( normal , initial wound , and chronic wound ) , and quantitative estimates for the relevant rate constants are provided . a definitive validation of the results , that is , the minimal kinetic model for predicting the beneficial effects of type a toxin , awaits additional clinical data . perhaps the most useful outcome is that our kinetic model is capable of identifying a measurable gap ( decay rate of toxin 's effect ) in our attempt to comprehend how complex , interacting biological systems respond to environmental stressors .
a relatively new approach in the treatment of specific wounds in animal models and in patients with type a botulinum toxin is the focus of this paper . the indications or conditions include traumatic wounds ( experimental and clinical ) , surgical ( incision ) wounds , and wounds such as fissures and ulcers that are signs / symptoms of disease or other processes . an objective was to conduct systematic literature searches and take note of the reactions involved in the healing process and identify corresponding pharmacokinetic data . from several case reports , we developed a qualitative model of how botulinum toxin disrupts the vicious cycle of muscle spasm , pain , inflammation , decreased blood flow , and ischemia . we transformed this model into a minimal kinetic scheme for healing chronic wounds . the model helped us to estimate the rate of decline of this toxin 's therapeutic effect by calculating the rate of recurrence of clinical symptoms after a wound - healing treatment with this neurotoxin .
You are an expert at summarizing long articles. Proceed to summarize the following text: the occurrence of neonatal seizures may be the first , and sometimes the only clinical sign of a central nervous system ( cns ) disorder in the newborn infant . neonatal magnetic resonance imaging ( mri ) is a relatively new technique which has rapidly become the study of choice for the evaluation of cns disorders in newborns . mri provides an excellent anatomical depiction of the brain which far surpasses cranial ultrasound and computed tomography . the advantage of mri is that it is the only technique which can identify myelination and differentiation of grey matter and white matter in the neonatal brain hence neonatal mri is becoming an important tool in predicting neurodevelopmental outcomes , and the future of mri is directed at understanding the prognostic implications of cns disease within newborns . after the introduction of mr compatible monitors and neonatal coils , safety and image quality has improved . according to alderliesten et al . , diffusion restriction ( dr ) changes in the mri is a good indicator of cell dysfunction ( reversible or irreversible ) within 1 week of insult . in a case series of perinatal stroke , dr changes in mri were strongly associated with motor sequelae . the main aim of this study was to find the association of dr changes in mri of the brain for neonatal seizures with long - term neurodevelopment outcome . retrospective data was collected for the time period from january 2010 to december 2011 from medical records department ( mrd ) for patient data , neonatal intensive care unit ( nicu ) and reports from picture archiving and communication system for mri images and the karthikeyan child development unit ( kcdu ) for their developmental follow - up reports . mrd in the hospital has the facility to retrieve case sheets with the diagnosis using international statistical classification of diseases and related health problems . with the search word of neonatal seizures - code no p-90 we found out 98 babies who had seizures within 28 days of life as shown in figure 1 . newborn babies who had seizures within 28 days of life and had undergone mri of the brain within 7 days of onset of seizures and who had undergone a developmental assessment at least at 6 months of corrected age were included in the study . babies with prematurity , congenital anomalies , and those who died flowchart of study group the standard unit protocol for neonatal seizures in our nicu is to do an mri of the brain within 7 days of onset of seizures . the various sequences taken were t1- and t2-weighted images and dr , mean corpuscular volume , mr angiogram for every baby . after explaining the procedure and obtaining parental consent , the babies were cuddled and well wrapped and during natural sleep usually postfeed if the baby is on feeding , the mri was done . the babies are sedated if needed with intranasal midazolam and monitored during the procedure with mri compatible monitors . the various mri abnormalities recorded included parenchymal infarction , dr in the ventrolateral thalamus and posterior putamen , dr in bilateral occipitoparietal cortex and splenium of corpus callosum , t1-flair hyperintensity , dysplastic splenium and corpus callosal thinning , hemorrhage in the lateral ventricle as shown in figure 2 . ( d ) occipital and splenium of corpus callosum changes suggestive of hypoglycemic encephalopathy all the babies in our institution with neonatal seizures were followed up by the kcdu for their developmental assessment at six and 12 months and then yearly for 6 years . as per the kcdu protocol , there would be a reminder letter sent before their scheduled follow - up , for those who do not report on that day , the second reminder through phone calls would be done , and the appointment would be rescheduled . for the study purpose , follow - up data of babies between 6 and 12 months of age were collected by matching with babies ' identity . fifty - seven out of the 98 babies who had undergone mri were matched with kcdu database ; there were 34 babies who had come for follow - up hence included in the study . complete data for 34 babies were collected , a wide range of parameters including maternal , perinatal , neonatal parameters such as gestational diabetes mellitus ( gdm ) , pregnancy - induced hypertension ( pih ) , seizure disorders , abnormal antenatal scan , mode of delivery , various resuscitative measures , gestational age , birth weight , head circumference , onset and type of seizure , and etiology of seizures . the developmental assessment was done using bayley 's scale of infant developmental tool iii at 6 and 12 months in the kcdu . if the child had come for both follow - up , then 12 months assessment was considered for the study analysis . composite for each domain is calculated . in this study , we considered delay in development when the neurological deficit is < 85% in any of the domain namely cognitive , language , motor , social , and adaptive [ table 1 ] . bayley scale of infant and toddler development tool ( ii ) comparison of a composite score for various domains with dr changes was analyzed with t - test and comparison of babies with developmental delay , and dr changes were analyzed with chi - square test . comparison of a composite score for various domains with dr changes was analyzed with t - test and comparison of babies with developmental delay , and dr changes were analyzed with chi - square test . in the present study , among the 98 babies who had neonatal seizures in the study period , 34 had both mri and developmental follow - up . twenty - nine had developmental follow - up at 1 year of age , 5 babies had come for follow - up at 6 months and lost to follow - up thereafter . among the study group , 23 ( 67% ) were male babies . the mean gestational age was 37 1.5 weeks ; birth weight of 2615 471.6 g and head circumference of 33 1.7 cm . gdm was found among 4 ( 11% ) and pih in 2 ( 6% ) of mothers . one ( 3% ) of them had abnormal umbilical doppler flow in the antenatal scan . six ( 17% ) had undergone emergency lower segment cesarean section ( lscs ) while 2 ( 6% ) had elective lscs . nine ( 26% ) received bag and mask ventilation and 3 ( 9% ) babies were intubated . the most common type of seizure is focal clonic in 18 ( 52% ) followed by multifocal in 9 ( 26% ) , subtle in 8 ( 23% ) , and generalized in 5 ( 14% ) and myoclonic seizures in 4 ( 11% ) . multiple types of seizures were present in 14 ( 41% ) babies , and none had tonic seizures . seizures occurred within 24 h in 12 ( 35% ) , between 24 and 72 h in 13 ( 38% ) and > 72 h in 9 ( 26% ) babies . the etiology of neonatal seizures with decreasing order of frequency based on clinico - radiological findings were hypoxic - ischemic encephalopathy ( hie ) 11 ( 32% ) , hypoglycemic encephalopathy 9 ( 26% ) , encephalitis 8 ( 23% ) , stroke 4 ( 11% ) and for hypocalcemia 2 ( 5.8% ) . table 2 depicts the etiological profile based on the clinic - radiological findings and dr changes . developmental delay was similar in both groups ( babies with dr changes and without dr changes ) . comparison of mri dr changes with developmental outcome in different domains namely cognition , language - receptive / expressive , fine and gross motor is shown in table 4 and there was no statistical significance among those who have dr changes and with those who do not have dr changes . etiological profile based on clinico - radiological findings and dr changes comparison of mri dr changes with developmental outcome comparison of mri dr changes with developmental outcome in different domains the topographic pattern of injury in the study series were parietal lobe changes in 14 , occipital lobe changes in 14 , frontal lobe changes in 5 , temporal lobe changes in 6 , internal capsule changes in 8 , corpus callosal changes in 9 , thalamus changes in 5 , cerebral hemispheres involvement in 7 , caudate nuclei in 2 , lentiform nuclei in 2 , right middle cerebral artery ( mca ) infarct in 1 , left mca infarct in 1 , hemorrhage in lateral ventricle in 2 . since the introduction of the diffusion - weighted image ( dwi ) , it has become an indispensible part of mri examination of the brain , as it is more likely to show the changes than the conventional mri , especially when done between the 2 and 4 day of seizure onset . dwi is a special sequence in mri of the brain which measures the alteration of the diffusion of water within the extracellular space and between intracellular and extracellular spaces . diffusion imaging complements conventional imaging in the evaluation of perinatal brain injury , as diffusion abnormalities better illustrate the extent of perinatal brain injury when compared with conventional mri , particularly when performed early . similarly , in our study , we obtained good delineation of the extent of perinatal brain injury . furthermore , because of the high water content of the neonatal brain , these changes were well marked on dwi . in our study , dr imaging yielded well as it was performed within 7 days , and it is known in literature that dr changes in certain etiology may disappear later as in hypoglycemia . however , there is insufficient evidence on the percentage of reversibility of dr changes in mri and its co - relation with the developmental outcome . a dr change is a marker of acute ischemia and is only one of the prognostic indicators . mri facilitates to define the various topographic pattern of injury , and we obtained dr changes mainly in the parietal lobe , occipital lobe , corpus callosum and internal capsule . due to nonavailability of bedside electroencephalography ( eeg ) , seizures were diagnosed clinically whenever there was a paroxysmal event otherwise unexplained . the most common type of seizures in our study is the clonic type ( 52% ) , and this type is known to have better eeg co - relation . babies ' neurodevelopmental outcome was assessed using bayley 's scale of infant and toddler development , this tool is an individually administered instrument which is one of the good tools to assess the developmental outcome between 1 and 42 months . the bayley - iii assesses infant and toddler development across five domains which are cognitive , language , motor , social , and adaptive . though the mean composite score is slightly less in the babies who had dr changes but it is not statistically significant . the seizure outcome would depend on the underlying etiology and the conditions with good prognosis ( 90100% normal ) are hypocalcemia and subarachnoid hemorrhage . conditions with poor prognosis ( < 50% normal ) are developmental defects , meningitis , hypoglycemia , hie . in our study , 11 out of 34 ( 32% ) were due to hie and 8 out of 34 ( 23% ) due to encephalitis . twenty - seven out of 34 ( 79% ) babies had seizures which were due to a condition which can lead to the potentially poor neurodevelopmental outcome . in hie , among 8 babies who had dr changes , 6 ( 75% ) had dd and out of the total 11 babies , 8 ( 73% ) had dd in one or more domain with a composite score of < 85% . in babies with encephalitis among 6 babies who had dr changes 4 ( 66% ) had dd . among the 2 babies who had hypocalcemia , no 1 had dr changes in mri but 1 ( 50% ) baby had dd . usually , hypocalcemic seizures are associated with good prognosis . in a study by ali et al . 26 out of 81 cases were found to have a developmental delay with normal mri of the brain , just as few cases of this indexed study . de vries et al . showed that increased signal intensity on dwi at the level of the posterior limb of internal capsule and cerebral peduncle in babies with stroke had subsequent hemiplegia . similarly , in our study , for stroke dr changes in mri is correlating well with neurodevelopmental outcome because all the babies with stroke had dr changes and developmental delay in gross motor , fine motor , and cognition . though dr changes may not be helpful to predict adverse long - term neurodevelopment outcome for these babies , it is found it has a better co - relation for babies with stroke . hie is known to affect the central gray nuclei and perirolandic cortex along with basal ganglia and brainstem as hyperechogenic and edematous changes ; a similar depiction was found in our study . in hypoglycemic encephalopathy sub group , they commonly have occipital lobe abnormalities , and , therefore , regular ophthalmological follow - up and visual assessment is very important among these babies as we can anticipate a problem with vision . cns infections usually impact the subcortical area , and site of the lesion may also vary depending on the organism causing the infection . group b streptococcus infection can represent ischemic changes in basal ganglia and thalamus while hydrocephalus and isolated dilatation of the fourth ventricle is seen with escherichia coli infection . streptococcus pneumonia and hemophilic influenza cause a subdural effusion . in expressive and receptive language domain , the mean composite score in both groups were above 88% which could be because there were done at 6 and 12 months . regarding fine motor domain , group with dr changes and without dr changes had low composite score in contrary to gross motor domain which had a composite score of > 85% in both groups . second , the sample size was small and few babies were lost to follow - up . we also did not have a follow - up mri of the study group to look for the persistence of the dr changes and development of t1- and t2-weighted changes in mri . though dr changes in mri may not predict the adverse long term neurodevelopmental outcome , but can be of use with regards to individual etiological profile as in stroke . larger group study and long - term follow - up are required to substantiate these findings . that dwi is a recommended imaging in babies with neonatal seizures is already known . our study adds that dr changes may not influence the adverse long - term outcome for these babies , but it can be of use with regards to individual etiological profile as in stroke .
background : neonatal seizures are a common manifestation of brain dysfunction . neonatal magnetic resonance imaging ( mri ) has rapidly become the study of choice for the evaluation of central nervous systems disorders in newborns . according to a study conducted in wilhelmina children 's hospital , university medical center utrecht , diffusion restriction ( dr ) changes in the mri is a good indicator of cell dysfunction ( reversible or irreversible ) within one week of insult.objectives:the main aim of this study was to find the association of dr changes in mri of brain for neonatal seizures with long term neurodevelopment outcome.methods:this is a retrospective observational study conducted in sri ramachandra university . retrospective data was collected for the time period of january 2010 to december 2011 from medical records department ( mrd ) for patient data , neonatal intensive care unit and reports from pacs for mri images and the karthikeyan child development unit for their developmental follow up reports.results:comparison of composite score for various domains with dr changes was done with a t - test and comparison of babies with developmental delay and dr changes with chi - square test . mri dr changes with developmental outcome in different domains namely cognition , language - receptive / expressive , fine and gross motor was studied . there is no statistical significance among those who have dr changes and with those who do not have dr changes.conclusion:though diffusion restriction changes in mri may not predict adverse long term neuro developmental outcome , they can be of use with regards to individual etiological profile as in stroke . larger group study and long term follow up is required to substantiate these findings .
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Proceed to summarize the following text: overactive bladder ( oab ) syndrome is described as " urgency , with or without urgency incontinence , usually with frequency and nocturia " by the international continence society ( ics ) terminology committee . however , a variety of medical conditions share the symptoms of oab , so it is important to rule out these causes . thus , the ics terminology committee stated that the term oab can be used only if there is no proven infection or other obvious pathology . urinary tract infection ( uti ) is the most common condition that can cause oab symptoms . the isolation of causative organisms through urine culture plays a crucial role in the diagnosis of uti . however , even if ordinary bacteria are not cultured , there is some evidence to suggest that atypical organisms , such as mycoplasmas , may be associated with oab symptoms [ 2 - 4 ] . mycoplasmas are the simplest microorganisms and lack a cell wall , a feature largely responsible for their lack of a reaction to gram stain and their lack of susceptibility to drugs such as penicillin that interfere with cell wall synthesis . although scientists have isolated at least 17 species of mycoplasmas from humans , four types are responsible for clinically significant infections . these species are mycoplasma pneumoniae , mycoplasma hominis ( m. hominis ) , mycoplasma genitalium , and ureaplasma species . these were generally viewed as commensals , although they usually manifest a predilection for particular host tissues , such as urogenital or respiratory tracts . however , it is now being recognized that these organisms play a more important role in human infections than was previously thought . their slow - growing , non - culturable nature enables them to establish chronic infections , resist the effects of antibiotics , and protect the organisms against immune system reactivity . accordingly , for patients presenting with chronic irritative urinary symptoms , especially those for whom ordinary cultures are negative , further tests can be indicated specifically for mycoplasmas . herein , we conducted a prospective study to estimate the incidence of genitourinary mycoplasma infections and to evaluate the efficacy of antibiotic treatment for these organisms in women with oab symptoms . the institutional review boards of the 4 study centers approved this study . written informed consent was obtained from all participants . inclusion criteria were women aged 20 and 80 years with micturition episodes 8/24 hours and urgency episodes 1/24 hours on a 3-day bladder diary . urgency episodes were defined as the number of micturition episodes associated with a urinary sensation scale rating 3 . exclusion criteria were a positive urine culture for bacteria using gram stain , postvoid residual 150 ml , history of pelvic irradiation , neuropathic bladder , and evidence of chronic inflammation such as interstitial cystitis . women who were pregnant or who had childbearing potential and did not use a reliable method of birth control during the study period were excluded . patients who required concomitant administration of the following drugs or treatment modalities were also excluded : anti - cholinergics , alpha - blockers , tricyclic antidepressants , selective serotonin reuptake inhibitors , electrical or electromagnetic stimulation , and neuromodulation . at the baseline visit , all participants were evaluated for their medical history , underwent a physical examination including pelvic examination , and completed the patient 's perception of bladder condition ( ppbc ) and international consultation on incontinence questionnaire - female lower urinary tract symptoms ( iciq - fluts ) . also , urethral and cervical vaginal swabs were obtained from all participants . the mycoplasma ist2 kit ( biomerieux , inc . , marcy - i'etoile , france ) was used for the culture , identification , and antibiotics susceptibility test for m. hominis and ureaplasma urealyticum ( u. urealyticum ) . colony forming units 10/ml were regarded as a positive result for m. hominis and u. urealyticum infection . for the detection of chlamydia trachomatis ( c. trachomatis ) , polymerase chain reaction ( pcr ) was used , and a positive result was regarded as infection . four weeks after negative conversion , patients were evaluated by use of a 3-day bladder diary , the ppbc , the iciq - fluts , and patient perception of treatment benefit questionnaire ( pptb ) . primary endpoints were the percentage of positive cultures for m. hominis , u. urealyticum , and c. trachomatis , and the change in the number of micturition episodes after negative conversion . secondary endpoints were the changes in the number of urgency episodes and changes on the ppbc , iciq - fluts , and pptb . for patients with persistent infection after treatment , the bladder diary , ppbc , iciq - fluts , and pptb were assessed 4 weeks after treatment . women with a positive result were initially treated with a 1 g dose of azithromycin . two weeks after treatment , follow - up swab samples were taken from the previously positive site , either the urethra or the cervical vagina . patients who had obtained negative conversion underwent no further treatment , whereas those with persistent infection were re - treated with 100 mg of doxycycline twice daily for 7 days , followed by additional swabs 2 weeks after the treatment . identical treatment was given to the patients ' sexual partners , and condom usage was recommended during the study period . potts et al showed that the difference in the mean number of micturition episodes was about -2.0/24 hours with a standard deviation of 3.5 after treatment of mycoplasmas in women with chronic urinary symptoms . with a 0.05 significance level and 80% power to detect a difference in the number of micturition episodes of -2.0/24 hours , a sample size of 27 patients was needed to be positive for infection . assuming a dropout rate of 20% and an incidence of mycoplasma infections in oab patients of 20% , 160 women with oab symptoms were scheduled to be screened . statistical analysis was performed by using the spss 17.0 ( spss , chicago , il , usa ) program for windows ( microsoft corporation , seattle , wa , usa ) . inclusion criteria were women aged 20 and 80 years with micturition episodes 8/24 hours and urgency episodes 1/24 hours on a 3-day bladder diary . urgency episodes were defined as the number of micturition episodes associated with a urinary sensation scale rating 3 . exclusion criteria were a positive urine culture for bacteria using gram stain , postvoid residual 150 ml , history of pelvic irradiation , neuropathic bladder , and evidence of chronic inflammation such as interstitial cystitis . women who were pregnant or who had childbearing potential and did not use a reliable method of birth control during the study period were excluded . patients who required concomitant administration of the following drugs or treatment modalities were also excluded : anti - cholinergics , alpha - blockers , tricyclic antidepressants , selective serotonin reuptake inhibitors , electrical or electromagnetic stimulation , and neuromodulation . at the baseline visit , all participants were evaluated for their medical history , underwent a physical examination including pelvic examination , and completed the patient 's perception of bladder condition ( ppbc ) and international consultation on incontinence questionnaire - female lower urinary tract symptoms ( iciq - fluts ) . also , urethral and cervical vaginal swabs were obtained from all participants . the mycoplasma ist2 kit ( biomerieux , inc . , marcy - i'etoile , france ) was used for the culture , identification , and antibiotics susceptibility test for m. hominis and ureaplasma urealyticum ( u. urealyticum ) . colony forming units 10/ml were regarded as a positive result for m. hominis and u. urealyticum infection . for the detection of chlamydia trachomatis ( c. trachomatis ) , polymerase chain reaction ( pcr ) was used , and a positive result was regarded as infection . four weeks after negative conversion , patients were evaluated by use of a 3-day bladder diary , the ppbc , the iciq - fluts , and patient perception of treatment benefit questionnaire ( pptb ) . primary endpoints were the percentage of positive cultures for m. hominis , u. urealyticum , and c. trachomatis , and the change in the number of micturition episodes after negative conversion . secondary endpoints were the changes in the number of urgency episodes and changes on the ppbc , iciq - fluts , and pptb . for patients with persistent infection after treatment , the bladder diary , ppbc , iciq - fluts , and pptb were assessed 4 weeks after treatment . women with a positive result were initially treated with a 1 g dose of azithromycin . two weeks after treatment , follow - up swab samples were taken from the previously positive site , either the urethra or the cervical vagina . patients who had obtained negative conversion underwent no further treatment , whereas those with persistent infection were re - treated with 100 mg of doxycycline twice daily for 7 days , followed by additional swabs 2 weeks after the treatment . identical treatment was given to the patients ' sexual partners , and condom usage was recommended during the study period . potts et al showed that the difference in the mean number of micturition episodes was about -2.0/24 hours with a standard deviation of 3.5 after treatment of mycoplasmas in women with chronic urinary symptoms . with a 0.05 significance level and 80% power to detect a difference in the number of micturition episodes of -2.0/24 hours , a sample size of 27 patients was needed to be positive for infection . assuming a dropout rate of 20% and an incidence of mycoplasma infections in oab patients of 20% , 160 women with oab symptoms were scheduled to be screened . statistical analysis was performed by using the spss 17.0 ( spss , chicago , il , usa ) program for windows ( microsoft corporation , seattle , wa , usa ) . of a total of 84 women screened , 42.8% ( 36/84 ) were found to be positive : 40.5% ( 34/84 ) for u. urealyticum , 7.1% ( 6/84 ) for m. hominis , and 3.6% ( 3/84 ) for c. trachomatis . about 8.3% ( 7/84 ) of women had 2 organisms : 7.1% ( 6/84 ) were positive for u. urealyticum and m. hominis and 1.2% ( 1/84 ) were positive for u. urealyticum and c. trachomatis . overall , 7 of the 36 positive women were excluded : 5 because of having taken anti - cholinergics and 2 because of being lost to follow - up . the demographic data and baseline characteristics of the 29 women are summarized in the table 1 . after treatment with antibiotics , 24 of 29 ( 82.7% ) patients obtained negative conversion : 14 ( 48.3% ) after the first treatment with azithromycin and 10 ( 34.5% ) after the second treatment with doxycycline . among 24 women with negative conversion , the median number of micturition episodes per 24 hours was reduced from 10.6 ( range , 8.0 - 19.3 ) to 8.1 ( range , 4.0 - 20.7 ) ( p=0.002 ) . the median number of urgency episodes per 24 hours was reduced from 2.7 ( range , 10.0 - 17.0 ) to 0.3 ( range , 0 - 20.7 ) ( p=0.097 ) . in terms of ppbc , the median score was improved from 4.0 ( range , 3.0 - 6.0 ) to 2.0 ( range , 1.0 - 6.0 ) ( p=0.001 ) , and the scores for 58.3% ( 14/24 ) of the women were improved by at least 2 points . in terms of the iciq - fluts , the filling and quality of life domain scores improved significantly ( fig . twenty - one of 24 ( 87.5% ) patients reported to have benefitted from the treatment and 9 ( 37.5% ) of them reported " much benefit " . also 21 of 24 ( 87.5% ) reported that they were satisfied with the treatment and 8 ( 33.3% ) of them reported that they were " very satisfied " . for the 5 women with persistent infection after antibiotic treatment , the median number of micturition episodes per 24 hours was reduced from 9.3 ( range , 8.0 - 14.3 ) to 8.3 ( range , 6.0 - 15.0 ) ( p=0.269 ) , and the median number of urgency episodes was reduced from 3.3 ( range , 1.0 - 9.3 ) to 0.0 ( range , 0.0 - 8.0 ) ( p=0.080 ) . four of the five ( 80% ) women reported having benefitted and one ( 20% ) of them reported " much benefit " from the treatment . four of five ( 80% ) reported that they were satisfied with the treatment and none of them reported that they were " very satisfied " . mycoplasma infection became a subject of interest in the early 1980s for its association with premature rupture of membranes , premature labor , endometritis , stillbirth , and neonatal respiratory distress syndrome . several studies have attempted to implicate mycoplasma infection as a cause of chronic urinary symptoms [ 2 - 4 ] . our results demonstrated that 42.8% of women with oab symptoms were positive for mycoplasma and chlamydia infections . the wide ranges are due to differences in the method of isolation ( e.g. , culture , serological assay , pcr , antigen detection enzyme - linked immunoabsorbant assay ) , method of sample collection ( e.g. , urine , cervical / vaginal swab , vaginal discharge ) , geographical and economical factors , and social and sexual habits . wave iii of the national longitudinal study of adolescent health tested 14,322 young men and women for mycoplasma infection with urine specimens . the prevalences of mycoplasma genitalium ( m. genitalium ) and chlamydia were 1.0% and 4.2% , respectively . the prevalence was 11 times higher among respondents who reported living with a sexual partner , and increased by 10% for each additional sexual partner . a community - based study reported an extremely high rate of mycoplasma infection ( > 70% for m. hominis and > 78% for u. urealyticum ) in papua new guinea . the authors explained that both low socioeconomic status and experience with multiple sexual partners were associated with the high rates . according to a retrospective comparative analysis of vaginal and endocervical cultures from 27,172 women visiting a genital tract diseases clinic , m. hominis was positive in 0.1% of symptomatic and 0.05% of asymptomatic women of childbearing age . this study reported that hormonal contraception and consistent condom use were protective factors for mycoplasma infections . in central / west and north africa , no steady partners , more than 1 partner in the previous 6 months , and intrauterine devices were risk factors . the presence of symptoms was not significantly related to the mycoplasma infections . on the other hand , another study showed different prevalence rates according to the presence of symptoms . among symptomatic women in a sexually transmitted disease clinic , m. genitalium and c. trachomatis were positive in 6% ( 26/461 ) and 10% ( 45/465 ) . however , the prevalence was 2% ( 1/59 ) and 0% , respectively , among women at a cancer screening center . a japanese study also showed 6.8% ( 8/117 ) of women with genital symptoms were positive for mycoplasma infections , compared with none of 80 women without symptoms . putting those various reports together , our prevalence rate was higher than those reported in the above studies , but is similar to that of potts et al who reported that 48% ( 23/48 ) of women with chronic urinary symptoms were positive for mycoplasmas ( 1 for m. hominis , 22 for u. urealyticum ) , although the inclusion criteria and the detection method differed from ours . a recent study also revealed high prevalence rates of mycoplasma infections using the mycoplasma ist2 kit , which was the same method that we used . they reported that about 53% ( 81/153 ) of women with chronic voiding symptoms were positive for mycoplasmas ( 5 for m. hominis , 81 for u. urealyticum ) . the individual infection rate of m. hominis and u. urealyticum was 7.1% and 40.5% , respectively , in our study . m. hominis infection was always associated with u. urealyticum infection . in the latter study , high rates of co - infection of m. hominis with u. urealyticum were also reported in the above - mentioned prevalence studies . this can be explained by the fact that m. hominis and u. urealyticum are important opportunistic pathogens in the genitourinary tract . after antimicrobial treatment , 24 of 29 ( 82.8% ) infected women obtained negative conversion . for the 5 women with persistent infection , susceptible antibiotics were given on the basis of the result of a follow - up test after the last assessment . four weeks after negative conversion , micturition frequency and scores on the symptom questionnaires were significantly improved , and the patient perception of treatment benefit and satisfaction was high . in other studies , micturition frequency and symptom severity were also improved after antimicrobial therapies . however , we failed to prove significant improvement in urgency episodes and some domain scores of the iciq - fluts questionnaire the other studies included women with a history of previous treatment for their symptoms . however , most of our patients ( 83% ) had no history of treatment . therefore , our patients might have had milder symptoms than in other studies . apart from the patient characteristics , we used a voiding diary for quantitative assessment of urgency episodes and standardized questionnaires , which differed from the other studies , in which a symptom severity score was applied , with 0 points indicating mild or no symptoms and 3 points indicating severe symptoms . these might also be why the improvement in urgency episodes and some domain scores on the questionnaire was not significant . burkhard et al evaluated the efficacy of antimicrobial treatment ( doxycycline ) for urinary urgency , frequency , and chronic urethral and pelvic pain in women . after treatment , 71% of the women became symptom - free or had a subjective decrease in symptoms . although causative organisms were not identified in most cases , the authors said that doxycycline was a broad - spectrum antibiotic that was effective against some of the most common bacterial causes of sexually transmitted diseases , such as mycoplasmas and chlamydia . another limitation was that , though the improvement did not reach statistical significance , there was a considerable improvement in symptoms among the 5 women with persistent infection . we can not explain this change , because there was no placebo group , but a placebo effect may be at least partially responsible . to confirm the causality of mycoplasma infections for oab symptoms and the efficacy of antimicrobial treatment for the symptoms , . this may not reflect practice in real life , in which anticholinergics are the first - line treatment for oab . therefore , study with patients who have persistent oab symptoms after anticholinergic medication would add more clinically relevant information on this subject . after antimicrobial treatment , micturition frequency and symptom questionnaires were significantly improved , and the patient perception of treatment benefit and satisfaction were high . diagnostic tests and treatment for genitourinary mycoplasmas would be beneficial for improving oab symptoms in women before invasive and costly diagnostic tests and treatments are considered .
purposeto evaluate the incidence of genitourinary mycoplasmas and the efficacy of antibiotics in women with overactive bladder ( oab ) symptoms.materials and methodswomen with oab symptoms ( micturition 8/24 hours and urgency 1/24 hours ) for 3 months were screened for mycoplasma hominis ( m. hominis ) , ureaplasma urealyticum ( u. urealyticum ) , and chlamydia trachomatis ( c. trachomatis ) . specimens from urethral and cervical vaginal swabs were examined for m. hominis and u. urealyticum by using the mycoplasma ist2 kit and for c. trachomatis by using pcr . women with positive results were treated with a 1 g dose of azithromycin . persistent infection was treated with doxycycline . changes in a 3-day bladder diary , patient perception of bladder condition ( ppbc ) , and international consultation on incontinence questionnaire - female lower urinary tract symptoms ( iciq - fluts ) were evaluated 4 weeks after negative conversion . patient satisfaction was assessed.resultsof 84 women screened , 42.8% were positive ( u. urealyticum , 40.5% ; m. hominis , 7.1% ; c. trachomatis , 3.6% ; two organisms , 8.3% ) . after treatment , 82.7% obtained negative conversion , and their median number of micturition episodes decreased from 10.6/24 hours to 8.1/24 hours ( p=0.002 ) . ppbc and domain scores of the iciq - fluts ( filling and quality of life ) significantly improved . about 87.5% women with negative conversion were satisfied with the treatment.conclusionsconsidering diagnostic tests and treatment for genitourinary mycoplasmas might be beneficial before invasive workup or treatment in women with oab symptoms .
You are an expert at summarizing long articles. Proceed to summarize the following text: coordinating egg donor in vitro fertilization ( ivf ) cycles with their recipients is vital to a successful reproductive outcome . synchronization of the donor and recipient menstrual cycles has historically been accomplished with the use of various formulations of combined oral contraceptive pills ( ocps ) ( 1 , 2 ) . however , this strategy relies on the donor 's responsibility to remember daily administration of pills . each year , more than three million women in the united states experience an unintended pregnancy , and almost half occur in women who reported using contraception ( 3 ) . although ocps are one of the most widely used contraceptive methods , patients experience difficulty in proper use of this medication . more than one million unintended pregnancies result from ocp method failure , misuse , and discontinuation ( 4 ) . in addition , the largest number of these unintended pregnancies occur among women aged 20 to 24 years ( 3 ) . for these reasons , a longer lasting contraception that may be administered in the office setting , such as the combined hormonal contraceptive vaginal ring , may be more optimal for the young egg donor population as it may take several months to prepare for and coordinate donor and recipient treatment cycles . large - scale studies have previously demonstrated the efficacy , tolerability , and acceptability of the combined contraceptive vaginal ring , and reliable cycle control with a low daily dose of ethinyl estradiol ( 59 ) . several studies have suggested that cycle control with the combined contraceptive vaginal ring was superior to that with a combined ocp ( 1012 ) . this attribute may be the result of local hormone administration and concentrations or stable hormone levels from continuous dosing . large efficacy trials have also shown that the ring is comparable with other combined hormonal contraceptive methods , with pregnancy rates less than one percent ( 57 ) . however , the ability of the combined contraceptive vaginal ring to obtain sufficient hypothalamic - pituitary - ovarian ( hpo ) axis suppression for ivf cycle synchronization with egg donor recipients has not been previously described . the objectives of this study were to evaluate the effects of the combined contraceptive vaginal ring on hpo axis suppression in egg donation ivf cycles as assessed by vaginal ultrasound imaging and serum gonadotropin levels . reproductive outcomes of the egg donation ivf cycles , including live birth rates , were also studied . we conducted a retrospective cohort study of all patients from the center for assisted reproduction ( care ) in bedford , texas undergoing in vitro fertilization cycles as egg donors from january 1st , 2003 through december 31st , 2009 . this time frame was chosen because care transitioned from using combined ocps to combined contraceptive vaginal ring for egg donor ivf cycles in 2006 . ninety - nine women participated in initial cycles as egg donors during the seven year time period , and all of these women were considered for our study in an effort to reduce selection bias . eight women were excluded from the study due to ivf monitoring outside of our facility . an additional two women were excluded because they were treated with the ortho evra patch ( janssen , antwerp , belgium ) and intramuscular depot provera prior to initiation of gonadotropins . of the remaining 89 patients , 45 women were treated with the combined contraceptive vaginal ring ( nuvaring ; merck , new jersey , u.s . ) and 44 women received various combined ocps prior to the initiation of gonadotropins . unfortunately , archived serum samples were unavailable for six of the women using the combined contraceptive vaginal ring and 19 women using the combined ocps . , 39 women who received the combined contraceptive vaginal ring and 25 women who received combined ocps were included in the study . institutional review board approval at the university of texas southwestern medical center was obtained for our research study . combined ocps administered to the patients included the following : desogestrel 0.15 mg / ethinyl estradiol 0.03 mg , desogestrel 0.15 mg / ethinyl estradiol 0.02 mg , desogestrel 0.15 mg / ethinyl estradiol 0.03 mg , ethynodiol diacetate 1 mg / ethinyl estradiol 0.035 mg , drospirenone 3 mg / ethinyl estradiol 0.03 mg , norgestimate 0.25 mg / ethinyl estradiol 0.035 mg and norethindrone 1 mg / ethinyl estradiol 0.05 mg . only the combined ocps with active medication were taken in preparation for the ivf process . the combined contraceptive vaginal ring administered in the office was the nuvaring ( merck , new jersey , u.s . ) . nuvaring is a flexible vaginal ring with an outer diameter of 54 mm and a cross - section of 4 mm . the ring releases 120 g of etonogestrel and 15 g of ethinyl estradiol per day , and insertion should occur between day one and five of the menstrual cycle . the contraceptive ring is designed for use during one cycle , comprising a three week period of continuous use followed by a one week ring - free period . therefore , when necessary , a new vaginal ring was administered every three weeks in the women using the combined contraceptive vaginal ring of this study . the combined contraceptive vaginal ring or combined ocps were used for a minimum of two weeks until the day prior to gonadotropin initiation . the gonadotropin medications used during the treatment cycles included follitropin beta injection ( follistim ; merck , new jersey , u.s . ) , follitropin alpha injection ( gonal - f ; emd serono , geneva , switzerland ) , and/or menotropins for injection ( menopur ; ferring , saint - prex , switzerland ) . ultrasonographic and laboratory evaluation was conducted at the care , and all decisions were made by three senior physicians . final follicle maturation was triggered with either hcg ( novarel ; ferring , saint - prex , switzerland ) or a gnrh agonist ( lupron ; abbott , illinois , u.s . ) . ultrasound - guided oocyte retrievals were performed under anesthesia approximately 36 hr following novarel or lupron administration . although the majority of embryos were cyropreserved on day 5 , cryopreservation of the remaining embryos was performed on days 5 through 7 . the electronic medical record at the care was utilized to obtain the majority of data for the study , including history and physical exams , ultrasound reports , laboratory reports , and ivf flow - sheets . previously stored blood samples drawn prior to gonadotropin initiation were analyzed onsite at the care for evaluating serum follicle - stimulating hormone ( fsh ) and luteinizing hormone ( lh ) levels . statistical analyses were performed using the sigmastat software package ( systat , chicago , il ) . numerical data were analyzed for normality followed by either the t - test or mann - whitney test , and categorical data were analyzed using either the chi - square test or fisher 's exact test . patient characteristics , including age , parity , race , body mass index , menstrual cycle length , and ovarian volume on ultrasound , were compared between the combined contraceptive vaginal ring and combined ocp groups ( table 1 ) . patient characteristics for comparing two methods of hpo axis suppression in egg donation ivf cycles prior to gonadotropin initiation , the endometrial thickness and serum estradiol were 5.62.6 mm and 33.619.9 pg / ml in the ocp group and 6.0 2.4 mm and 36.624.3 pg / ml in the combined contraceptive vaginal ring group , respectively ( p=0.49 and p=0.33 ) . average serum fsh and lh were 1.71.9 and 1.72.5 miu / ml in the ocp group and 1.71.6 and 1.21.4 miu / ml in the combined contraceptive vaginal ring group , respectively ( p=0.45 and p=0.95 ) . no significant differences were found for gonadotropin requirement , peak estradiol , maximal endometrial thickness , number of oocytes retrieved , number of normally fertilized embryos , or number of cryopreserved embryos ( table 2 ) . the average numbers of embryos transferred to the egg donor recipients during the fresh ivf cycle were 1.80.4 and 1.90.4 ( p=0.31 ) in the ocp group and combined contraceptive vaginal ring group , respectively . ivf cycle data of women in comparing two methods of hpo axis suppression in egg donation ivf cycles pregnancy outcomes in the egg donor recipients from fresh ivf cycles are shown in table 3 . there were no significant differences for any of the pregnancy outcomes , including live birth rates , between the ocp group and combined contraceptive vaginal group . the combined contraceptive vaginal ring has been shown to produce mean serum ethinyl estradiol concentrations of 19 pg / ml and maximum serum concentrations of 35 pg / ml ( 13 ) . pharmacokinetic evaluation revealed that after reaching maximum serum concentrations of etonogestrel and ethinyl estradiol , levels decreased linearly over time , in contrast to daily peaks of the combined ocp ( 13 ) . the maximum serum concentrations of etonogestrel and ethinyl estradiol were approximately 40 and 30 g , respectively , compared to the levels with a combined ocp containing 150 g desogestrel and 30 g ethinyl estradiol ( 13 ) . exposure to ethinyl estradiol from the combined contraceptive vaginal ring was on average 2.1 times lower than those using combined ocps containing 150 g levonorgestrel and 30 g ethinyl estradiol ( 14 ) . variations in individual serum ethinyl estradiol levels were far greater with either the combined ocp compared to the combined contraceptive vaginal ring ( 14 ) . a number of other studies have reported similar pharmacokinetic results ( 13 , 15 , 16 ) treatment with the combined contraceptive vaginal ring or combined ocp containing 150 g desogestrel and 20 g ethinyl estradiol resulted in comparable tissue concentrations of ethinyl estradiol and etonogestrel in samples from the upper myometrium , mid myometrium , and cervical region of the uterus ; however , the combined contraceptive vaginal ring group showed lower concentrations of both hormones in the endometrium ( 17 ) . therefore , the combined contraceptive vaginal ring is not associated with elevated uterine concentrations of ethinyl estradiol or etonogestrel compared with a combined ocp ( 17 ) . overall , differences exist between the pharmacokinetics of the combined contraceptive vaginal ring and combined ocps which could potentially result in variations between their treatment outcomes . the combined contraceptive vaginal ring has consistently been shown to have good cycle control in several large studies ( 5 , 7 , 10 , 11 ) . furthermore , cycle control with the combined vaginal contraceptive ring has been shown to be superior to that with a combined ocp containing 30 g ethinyl estradiol and 150 g levonorgestrel ( 10 , 11 ) or 3 mg drospirenone ( 12 ) . the incidence of irregular bleeding with the combined contraceptive vaginal ring is approximately five percent in all cycles ( 5 , 7 , 8 , 10 ) . with combined ocps , 15 g / day dosage of ethinyl estradiol results in irregular bleeding in approximately 29 percent of women during the initial six cycles ( 18 , 19 ) . continuous vaginal ring use also resulted in an acceptable bleeding profile in most patients with a reduction in flow , a reduction in pelvic pain , and a high continuation rate ( 20 ) . the good cycle control achieved with the combined contraceptive vaginal ring may be the result of the continuous release of steroids from the ring , avoiding daily fluctuations in the hormone levels . ovarian suppression achieved with the combined contraceptive vaginal ring is comparable to that observed with low - dose combined ocps ( 21 ) . during the normal three week period of use , the combined contraceptive vaginal ring completely inhibited ovulation as assessed by vaginal ultrasound and serum concentrations of fsh , 17 estradiol , lh , and progesterone ( 21 ) . extending its use for an additional two weeks did not compromise ovarian suppression and inhibition of ovulation was maintained ( 21 ) . irrespective of the length of a second cycle with the combined contraceptive vaginal ring ( three weeks versus three days ) , a new cohort of follicles needed to be recruited and the time to ovulation after ring removal was similar ( 19 versus 17 days ) ( 22 ) . the median time needed to develop a 13 mm follicle was 11 days , and none of these women ovulated after insertion of a second contraceptive ring ( 22 ) . therefore , ovulation , at least until the stage of a 13 mm follicle , is prevented and as little as three consecutive days of combined contraceptive vaginal ring use interferes with follicle growth ( 22 ) . in contrast , the generally acknowledged requirement for combined oral contraceptive pills to suppress the hpo axis is seven days ( 23 ) . although , in comparing the first cycle of combined contraceptive vaginal ring to combined ocps , there was a tendency towards slightly higher maximum follicular diameters , fsh , and lh levels in the combined contraceptive vaginal ring group ( 24 ) . however , the observed difference could be due to chance , differences in pharmacokinetic parameters , or differences in the timing of initiation ( combined contraceptive vaginal rings were inserted on cycle day five and combined ocps were initiated on cycle day one of menses ) ( 24 ) . women with shorter cycles and early ovulations in the pretreatment cycle developed larger follicles during treatment with the combined contraceptive vaginal ring compared to those with longer cycles and late ovulations ( 25 ) . overall , the combined contraceptive vaginal ring is a very effective and reversible method of hormonal contraception for women . combined contraceptive vaginal ring users experience many of the same side effects commonly described with the use of other combined hormonal contraceptives ( 26 , 27 ) ; however , the combined contraceptive vaginal ring has higher incidences of vaginal discomfort , vaginitis , and ring - related events ( 5 , 7 , 10 , 28 ) . although , a high level of user and partner acceptability for the combined contraceptive vaginal ring has been reported ( 5 , 7 , 2932 ) . 96 percent of women using the combined contraceptive vaginal ring for 13 consecutive cycles were satisfied with their method of contraception ( 29 ) . reasons for liking the combined contraceptive vaginal ring included not having to remember anything and ease of use ( 17 , 29 ) . a one - year , open - label , randomized controlled trial compared the safety of the combined contraceptive vaginal ring with a combined oral contraceptive containing 30 g ethinyl estradiol and 150 g levonorgestrel 6 or 3 mg drospirenone ( 9 ) . these studies demonstrated that the combined contraceptive vaginal ring exhibits tolerability and safety equivalent to that of an oral contraceptive ( 6 , 9 ) . our study is a retrospective study and the disadvantages include selection bias and information bias as well as the reliance on accurate record - keeping . it would be beneficial to pursue a prospective study in the future however , in an effort to minimize potential bias , all women were considered for inclusion in the study during the specified timeline and any exclusion was clearly defined . in addition , the electronic medical record at the care assisted in the retrieval of patient data . another disadvantage to our study is the transition from the use of combined ocps to the combined contraceptive vaginal ring in egg donor ivf cycles . clinical and laboratory protocols continuously evolve over time , and this could potentially alter the outcomes studied . effective contraception is important in preventing unplanned pregnancy in the selected egg donors preparing for the process of ivf . overall , studies have demonstrated that use of the combined contraceptive vaginal ring successfully inhibits ovulation with a good safety profile and high levels of patient satisfaction . however , there is a paucity of information regarding the utility of this contraceptive in achieving hpo axis suppression in egg donation ivf cycles . as assessed by transvaginal ultrasound and serum gonadotropin levels , we have now demonstrated that the combined contraceptive vaginal ring blunts activity of the hpo axis to a degree that is comparable to the use of ocps in egg donation cycles . in addition , both methods of contraception are associated with similar reproductive outcomes in ivf cycles , including live birth rates . thus , our data suggest that synchronization of the egg donor and embryo recipient cycles can be achieved effectively with either mode of hormonal delivery . nevertheless , the combined contraceptive vaginal ring provides a substantial benefit as it may be placed in the office and , therefore , does not depend on reliable daily self - administration of pills . improving compliance with medications during egg donation ivf cycles facilitates optimal outcomes for the recipients .
backgroundour study compares the efficacy of the combined contraceptive vaginal ring to oral contraceptive pills ( ocps ) for hypothalamic - pituitary - ovarian ( hpo ) axis suppression in egg donor in vitro fertilization ( ivf ) cycles.methodsour retrospective cohort study includes patients from the center for assisted reproduction ( care ) in bedford , texas undergoing ivf cycles as egg donors from january 2003 through december 2009 . twenty - five and thirty - nine women were treated with ocps and the combined contraceptive vaginal ring , respectively . statistical analyses were performed using the sigmastat software package ( systat , chicago , il ) . data were analyzed by t or mann - whitney test and chi - square of fisher exact test . statistical significance was set at p<0.05.resultsprior to gonadotropin initiation , endometrial thickness and serum estradiol were 5.62.6 mm and 33.619.9 pg / ml in the ocp group and 6.02.4 mm and 36.624.3 pg / ml in the combined contraceptive vaginal ring group , respectively ( p=0.49 and p=0.33 ) . average serum fsh and lh were 1.71.9 and 1.72.5 miu / ml in the ocp group and 1.71.6 and 1.21.4 miu / ml in the combined contraceptive vaginal ring group , respectively ( p=0.45 and p=0.95 ) . no significant differences were found for gonadotropin requirement , peak estradiol , maximal endometrial thickness , number of oocytes retrieved , number of normally fertilized embryos , number of cryopreserved embryos , or live birth rates.conclusionthe combined contraceptive vaginal ring is effective for hpo axis suppression in egg donor ivf cycles and associated with cycle characteristics similar to those observed with ocp treatment . the combined contraceptive vaginal ring may provide an important advantage over ocps due to improved patient compliance .
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Proceed to summarize the following text: the world health organisation estimates that each year more than 8 million new cases of tb occur and approximately 3 million people die of the disease worldwide . recently , the increase in the incidence of drug resistant tb has further worsened the problem . oral tb is rare and accounts for less than 1% of all cases of tb . with the increasing number of tb cases , unusual forms of the disease in the oral cavity a 7-year - old boy presented with difficulty in swallowing solid foods , low - grade fever and loss of weight for 11/2 month . there was no history of cough , abdominal pain , vomiting , diarrhea , or urinary complaints . on admission , his heart rate was 104/min , the respiratory rate was 28/min and blood pressure was 98/60 mmhg . his weight was 13 kg , and height was 104 cm ( both below the fifth percentile for age and sex ) . oral cavity examination showed perforation of the hard palate , 3 3 cm with an irregular , undermined margin , and a necrotic base [ figure 1 ] . investigations showed : hemoglobin 8.8 g / dl , total leucocyte count 9000/cumm ( neutrophils 40% , lymphocytes 60% ) , erythrocyte sedimentation rate 80 mm at the end of 1 h. his liver and renal functions tests were normal . computed tomography scan of the oral cavity showed erosion and scalloping of the posterior part of the hard palate on the right - side [ figure 2 ] . high power view of papanicolou stain of fine - needle aspiration of the lymph nodes showed granular caseous necrosis and granuloma composed of epitheliod cells and histiocytes [ figure 3 ] . however , in view of the clinical presentation and evidence of pulmonary and lymph node tb , the palatal perforation was most likely due to tb . he was started on antituberculous therapy ( isoniazid , rifampicin , pyrazinamide , and ethambutol ) and is on regular follow - up with the oral surgeons for repair of the palatal perforation . oral cavity examination showing perforation of the hard palate , 3 3 cm in size with an irregular , undermined margin , and a necrotic base plain axial computed tomography scan of midface with 3d reconstruction showing erosion and scalloping of the posterior part of the hard palate on the right side high power view of papanicolaou stain of fine needle aspiration of the cervical lymph nodes showing granular caseous necrosis and granuloma composed of epithelioid cells and histiocytes oral tb may either be primary , or more often , secondary to pulmonary tb . in secondary oral tb , the bacilli reach the oral mucosa by hematogenous or lymphatic spread . in primary oral tb there is direct inoculation of the mycobacterium due to break or loss of the natural barrier resulting from trauma , inflammatory conditions , leukoplakia , tooth extraction , or poor oral hygiene . other local predisposing factors include dental cysts , periapical granulomas , dental abscess , periodontitis , and jaw fractures.[24 ] abbot et al . were able to isolate the tubercle bacilli from mouth washings of 44.9% of the patients with active pulmonary lesions and thus highlighting the importance of an intact mucosal epithelium in resisting oral tb infection . the systemic factors that favor the chances of oral infection in tb includes lowered host resistance and increased virulence of the organisms . tuberculous involvement of oral cavity is rarely seen even in populations with high incidence of the pulmonary disease . factors that attribute to relative resistance of oral cavity to tb are protective effect of saliva , presence of saprophytes , resistance of striated muscles to bacterial invasion , and thickness of protective epithelial covering . other sites include the soft palate , hard palate , lip , cheek , tonsils , gingiva , floor of mouth , uvula , and alveolar mucosa . table 1 highlights some of the reported cases of oral tb in the literature.[2369 ] oral tb lesions usually manifest as single , non - healing ulcers with an indurated , irregular , undermined margin , and a necrotic base . oral lesions can also present as nodules , fissures , plaques , vesicles , tuberculomas , or granulomas . the palatal lesion of tb may be seen as granulomas , ulceration , or perforation and are usually more common in the hard palate than in the soft palate . according to baruah , et al . palatal involvement of tb resulting in perforation is usually seen in patients with strong immune responses and considered to be due to hypersensitivity to acid - fast bacilli that causes tissue destruction . reported cases of oral tuberculosis in addition to tb , the causes of palatal perforation include infection ( syphilis , leprosy , leishmaniasis , or fungal infection ) , wegener 's granulomatosis , sarcoidosis , neoplasms ( salivary or squamous cell ) , drug abuse ( cocaine ) , and midline lethal granuloma . in our case , the above differentials were unlikely in view of the clinical presentation , evidence of pulmonary and lymph node tb and the clinical improvement seen with antituberculous therapy on follow - up . the treatment of palatal tb should follow the general guidelines established for the treatment of extrapulmonary tb . tb of the palate is relatively rare and should be included in the differential diagnosis of palatal perforation . also , a search for the primary site should be done extensively in all the cases of palatal tb .
tuberculosis ( tb ) of the hard palate is rare and usually secondary to pulmonary tb . we present a case of a 7-year - old boy who presented with difficulty in swallowing solid foods , low - grade fever and loss of weight . oral cavity examination showed perforation of the hard palate with an irregular , undermined margin and a necrotic base . chest x - ray showed homogeneous opacity in the right upper zone . fine - needle aspiration of the cervical lymph nodes showed granular caseous necrosis and granuloma composed of epitheliod cells and histiocytes . in view of the clinical presentation and evidence of pulmonary and lymph node tb , the palatal perforation was most likely due to tb . patient was started on antituberculous therapy and is on regular follow - up .
You are an expert at summarizing long articles. Proceed to summarize the following text: prostate cancer is one of the most prevalent types of cancer in men , and transrectal ultrasound ( trus)-guided prostate biopsy , a reliable and easy - to - perform method , is accepted as the gold standard in the diagnosis of this disease . the use of prostate - specific antigen ( psa ) in clinical practice has led to an increase in the prevalence of prostate cancer . elevated serum psa levels or suspected prostate cancer on the rectal examination require prostate biopsy . in patients with negative initial biopsy results , some small foci of atypical glands with some features of adenocarcinoma are found in 1.5% to 9% of prostate biopsy specimens . asap , which is defined as a " focus of small acinar structures formed by atypical epithelial cells , " is a condition in which the pathologist has insufficient data to make a diagnosis and thus raises the suspicion of cancer . it has been reported that the cancer detection rate varies between 21% and 51% on the second biopsy in patients with asap . prostatic intraepithelial neoplasia ( pin ) is defined as " premalignant proliferation of atypical ductal and acinar cells . " high - grade prostatic intraepithelial neoplasia ( hgpin ) is reported in 5% to 7% of prostate biopsies . the risk of subsequent cancer varies between 25% and 79% . because the sextant biopsy technique has been increasingly replaced by multiple sampling methods , cancer detection rates on repeat biopsies following hgpin have declined . in clinical practice , asap and hgpin are two common findings observed on prostate biopsies . knowing the rate of prostate cancer diagnosis on repeat biopsies would aid primary treating physicians in the decision - making process for suspicious cases . for this purpose we retrospectively reviewed the medical records of 2,433 patients who had undergone trus - guided prostate biopsy for the diagnosis of prostate cancer between 2003 and 2009 at our hospital . the biopsy results of the patients were classified into four categories : prostate cancer , normal prostates or benign prostatic hyperplasia ( bph ) , asap , and hgpin . in the present study , 143 patients with asap or hgpin whose biopsy specimens had not revealed prostate cancer and who had undergone at least one repeat biopsy were enrolled . age , digital rectal examination ( dre ) findings , prostate volumes ( pv ) , free and total psa levels , and the biopsy results of the patients were recorded . the tissue samples were stained with racemase ( amacr ) , p63 , and high - molecular weight cytokeratin ( clone 34e12 ) in foci suspicious of cancer ( asap ) in an effort to make a definitive diagnosis of adenocarcinoma . the patients in whom repeat biopsies had not been performed for any reason or those having < 8 quadrant and extended initial biopsies were excluded from the study . the levels of psa and free psa , pvs , and dre findings of the patients with asap / hgpin on the initial biopsy were compared with respect to the presence of cancer . the pearson 's chi - square test was used to analyze differences in the cancer development rate for asap and hgpin . after the initial biopsy , prostate cancer was diagnosed in 742 of 2,433 patients ( 30.5% ) and normal prostates or bph was reported in 1,513 patients ( 62.2% ) . asap was found in 114 patients ( 4.7% ) , hgpin in 58 patients ( 2.4% ) , and asap+hgpin in 6 patients ( 0.2% ) ( table 1 ) . one hundred forty - three patients in whom repeat biopsy results were available were included in the study . of these patients , the mean psa was 11.3310.9 ng / ml , the mean free / total psa ratio was 0.310.73 , and the mean prostate volume was 60.232.4 cc . in 21 patients , dre findings were found to be significant regarding the presence of cancer ( table 2 ) . age , pv , and psa values did not show any statistically significant differences when patients reported as having asap and those reported as having hgpin were compared ( p>0.05 ) , whereas abnormal dre findings were significantly higher in the asap group . of the 143 patients , asap , hgpin , and asap+hgpin were detected in 97 , 40 , and 6 patients , respectively , in the initial biopsies . although second biopsies were performed in all patients , third biopsies were performed in only 27 patients with asap and 9 patients with hgpin . of the 97 patients with asap , prostate cancer was detected in the second and third biopsies of 32 and 6 patients , respectively . the gleason score was found to be 6 in 26 patients and 7 in 6 patients who were diagnosed as having prostate cancer after the second biopsy . all the patients who were diagnosed as having prostate cancer after the third biopsy had a gleason score of 6 . the total number of patients diagnosed as having prostate cancer was 38 , and 22 of them had t1c tumors , whereas 16 had t2a . none of the 40 patients with hgpin had evidence of prostate cancer in the second or third biopsy . of the 6 patients with asap+hgpin , prostate cancer was detected in 3 patients in the second biopsy and in 1 patient in the third biopsy ( table 3 ) . furthermore , on repeat biopsies , asap and hgpin were detected in 12 and 5 patients , respectively . the incidence of prostate cancer increases with age , and it ranks second to lung cancer in terms of incidence and mortality risk . significant advances have been achieved in the diagnosis and treatment of prostate cancer with the introduction of psa and prostate biopsy techniques . however , the histological diagnosis of prostate cancer in biopsy specimens remains a challenge for pathologists . a lack of availability of immunohistochemical staining , which may differentiate benign from malignant acini , and the poor diagnostic significance of the absence of basal cells in some cases complicate the diagnosis of prostate cancer . trus - guided prostate biopsy , which is a reliable and easy - to - perform method , is accepted as the gold standard in the diagnosis of prostate cancer . the purpose of the biopsy is to determine prostate cancer foci by obtaining a sufficient amount of samples at the true location without increasing morbidity . in the prostate biopsies performed for suspected prostate cancer , positive results confirm the diagnosis ; however , the suspicion of cancer remains with negative results . moreover , the suspicion of cancer becomes more prominent if the results of the biopsy reveal asap or hgpin . keetch et al reported cancer detection rates in 4 consecutive biopsies to be 34% , 19% , 8% , and 7% , respectively in a group of 1,136 patients with psa values higher than 4 ng / ml . in accordance with the literature , in the present study , four main patterns of hgpin have been identified as follows : flat , micropapillary , cribriform , and tufting . however , other unusual histologic types of hgpin , such as foamy gland , signet - ring cells , and mucinous have also been reported . borboroglu et al showed a 4% hpin rate in extensive repeat biopsies in a series of 57 cases . because the sextant biopsy technique has been increasingly replaced by multiple sampling methods , cancer detection rates on repeat biopsies following hgpin have declined . thus , repeat biopsy should be performed if hgpin is detected by a sextant biopsy technique , and if the biopsy has been performed with multiple sampling , other parameters , such as psa or rectal examination status , should be considered . in the present study , prostate cancer was not detected in any of the 40 patients with hgpin . recently , multiple sampling with laterally directed systematic sextant biopsies has been shown to increase the cancer detection rate by providing sufficient sampling in the first biopsy . thus , hgpin appears to be a precancerous lesion of prostate cancer . as with prostate cancer , the prevalence of hgpin also increases with age and represents a similar geographic / ethnic distribution . it is important to note that the detection of prostate cancer on repeat biopsies in patients with hgpin detected in their first biopsies is not a missed diagnosis in the initial biopsy , but in fact might reflect the development of a new cancer arising from hgpin . the rate of prostate cancer diagnosis in a 3 year follow- up among patients who were initially diagnosed as having hpin was 25.8% in a small group studied by lefkowitz et al . it is known that needle biopsy does not provide clear findings for the diagnosis of prostate cancer . although the structural and cytological features of a small number of glands in a small focus may have atypical appearances on needle biopsy , such findings may be insufficient for the diagnosis of adenocarcinoma . the terms asap , focal glandular atypia , atypical biopsy suspicious for malignancy , and borderline lesions are used in such conditions . the term asap was coined by iczkowski et al in 1997 ; however , it is not recommended by all pathologists . in a consensus meeting in 2004 sponsored by the world health organization , the reasons for this include the equation by some urologists of the term asap with hgpin , and because all of the atypical foci are not always " small " acinar but may include glands with a larger diameter ( such as a pseudohyperplastic pattern of cancer or adenocarcinoma with ductal features ) . indeed , asap is a condition in which the pathologist has insufficient data to make diagnosis , and thus raises the suspicion of cancer . the cancer detection rate on repeat biopsy is 21% to 51% , and a subsequent biopsy should be directed to the locations in which asap was found in the initial biopsy , because a significant portion of the cancers are located in these regions . in the present study , 38.2% of the patients shown to have asap in the initial biopsy were diagnosed with prostate cancer . therefore , the high percentage of prostate cancer in patients with asap in the initial biopsies makes the need for aggressive follow - up reasonable . the cancer detection rate declines with increasing prostate volume . levine et al reported that the cancer diagnosis rate was 43% , 27% , and 24% in men with prostate volumes less than 30 cc , between 30 cc and 50 cc , and greater than 50 cc , respectively . in second biopsies , two - fold higher positive results were obtained in patients with larger prostate volumes . it is now accepted that the standard sextant biopsy technique does not provide adequate sampling . hence , multiple sampling must be performed in patients with larger prostate volumes either in the initial or on repeat biopsies . although psa is specific to prostate tissue , it is not specific to prostate cancer . the positive predictive value of serum psa > 4.0 ng / ml is reported to be 25% for prostate cancer . when the cut off value is accepted to be 2.5 ng / ml , prostate cancer detection rates increase , but the number of unnecessary biopsies also increases . although the percent free psa maintains sensitivity for the early detection of prostate cancer , it also significantly improves the specificity of psa . a low percent free psa indicates an increased risk of cancer ; however , cancer is detected on biopsies of 30% to 50% of men with percent free psa levels < 15% . in the present study , we observed that the total psa and the percent free psa levels significantly increased the cancer detection rates in patients with asap in the initial biopsy . this condition is predictable for asap , which is associated with higher cancer detection rates on repeat biopsies . a limitation of the present study was that the biopsy samples were not examined by the same pathologist , although all biopsies were examined in the same pathology laboratory . furthermore , the retrospective design of the study and changes in psa threshold level over time might have influenced the homogeneous distribution of the study population . in the present study , the psa threshold value was 4.0 ng / ml in approximately one - half of the patients , whereas it was 2.5 ng / ml for the rest of the patients . a repeat biopsy should be performed for the entire prostate subsequent to the diagnosis of asap and the patients should be closely followed for the risk of cancer . if the initial biopsies are performed with multiple sampling , the final decision should be based on other parameters , such as psa values and rectal examinations in patients with a biopsy result of hgpin . we need to carefully follow - up such patients for their progression towards cancer in the future .
purposein clinical practice , atypical small acinar proliferation ( asap ) and high - grade prostatic intraepithelial neoplasia ( hgpin ) are two common findings on prostate biopsies . knowing the frequency of a prostate cancer diagnosis on repeat biopsies would aid primary treating physicians regarding their decisions in suspicious cases.materials and methodsone hundred forty - three patients in whom biopsies revealed asap or hgpin or both were enrolled in the present study ; prostate cancer was not reported in the biopsy specimens and at least one repeat biopsy was performed . age , digital rectal examination findings , prostate volumes , and free and total prostate - specific antigen ( psa ) levels and the biopsy results of the patients were recorded.resultsof the 97 patients with asap on the first set of biopsies , prostate cancer was diagnosed in the second and third biopsies of 32 and 6 patients , respectively . prostate cancer was not detected in the second or third biopsies of the 40 patients with hgpin in the first biopsy . of the 6 patients with asap+hgpin in the first biopsy , prostate cancer was detected in 3 patients in the second biopsy and in 1 patient in the third biopsy.conclusionsthe diagnosis of asap is a strong risk factor for prostate cancer . a repeat biopsy should be performed for the entire prostate subsequent to the diagnosis of asap . in patients with hgpin according to the biopsy result , the clinical decision should be based on other parameters , such as psa values and rectal examination , and a repeat biopsy should be avoided if the initial biopsy was performed with multiple sampling .
You are an expert at summarizing long articles. Proceed to summarize the following text: the first cohort comprised anonymized residues of serum samples submitted in 2000 for microbiologic or biochemical testing for diagnostic or screening purposes to 11 laboratories in england that were contributing to the public health laboratories service ( phls ) serologic surveillance programme ( now health protection agency [ hpa ] ) seroepidemiology programme ( 3 ) . the second cohort comprised 518 samples collected in 1995 from the phls cohort of farm workers , which was recruited in 1991 to provide annual samples from a population at high risk for zoonotic infections from livestock ( 4,5 ) . serum specimens were initially screened at a dilution of 1:10 in phosphate - buffered saline ( pbs ) , ph 7.2 , containing tween 20 ( pbs / tween ) with an inhibition elisa developed in our laboratory and previously validated in cattle and dogs ( 6 ) . positive and negative bovine serum controls were used on each elisa plate . in the absence of a human positive control , we used a primate serum sample as a further positive control . optical density was read at 450 nm , and percentage inhibition ( pi ) values were calculated by using the formula 100 [ ( test od / negative control od ) 100 ] . without specific validation , a cut - off of 20% inhibition was chosen to indicate putative positives . this cut - off has been used for previous comparison of the inhibition elisa with a conventional n. caninum all samples with inhibition > 20% were subsequently tested in an immunofluorescence test ( ifat ) ( 7 ) with appropriate species - specific fluorescein isothiocynate conjugates . all samples were tested at a dilution of 1:50 with positive controls of bovine and primate n. caninum serum and bovine negative control serum as above . for the inhibition elisa results , the distribution of the data was examined first by plotting percent inhibition , with the data aggregated into bands of 10% . the plots were repeated after logging the data and putting it into bins ( equal width reactivity categories based on log 10% [ log10 ] inhibition ) . the distribution of log10 inhibition was also examined according to person s sex and age and the submitting laboratory . from the hpa collection , 3,232 samples from persons 2070 years of age were tested ; 1,889 ( 58.45% ) were from women . the phls farm cohort , comprised 74% men and 26% women , with a median age range of 4150 years ( 5 ) . six hundred and ninety - one ( 21.38% ) of the hpa samples and 29 ( 5.56% ) of the phls samples produced percentage inhibition of > 20% in the inhibition elisa test ( table ) . for the bovine - positive controls , mean percentage inhibition was 79.76% ( 5% confidence interval [ ci ] 78.9480.58 , n = 200 ) . the primate - positive serum had a pi of 63.00% ( average of 2 tests ) . when samples with a pi > 20% in the inhibition elisa were tested by ifat , all failed to give positive fluorescence results , whereas the primate- and bovine - positive controls consistently gave positive fluorescence results . the frequency distribution of actual percentage inhibitions for the hpa samples showed a single positively skewed distribution . after the data were logged , the plot showed a single log - normal distribution , with a mean close to 0% inhibition , i.e. , similar to the negative control used . there was no evidence that the percentage inhibition differed by sex or region ( results not shown ) or by age ( figure ) . the log - transformed inhibition data for the phls cohort also produced a single normal frequency distribution about a mean close to 0% inhibition ( figure ) . frequency distribution of inhibition elisa results for neospora caninum , england ( health protection agency serum samples ) , stratified by age group . * equal - width bands based on log10 percentage inhibition . in this study , we sought evidence of human exposure to n. caninum infection by specific antibody detection in 2 populations . since no serologic assay has been validated for n. caninum antibodies in humans , we used an inhibition elisa and analyzed the frequency distribution of the percentage inhibitions . although initial screening gave several putative positives , when the frequency distribution curves were plotted , no evidence was found showing that the samples were distributed discretely into those with antibody to neospora spp . and those without . this provides strong evidence that no neospora - specific immunoglobulin g was found in any of the samples and that the distribution observed describes a single population of nonexposed persons . moreover , no change in distribution was observed with age , further evidence that the dataset is from a single large population for which results were negative . finally , confirmatory testing with the ifat of all samples with > 20% inhibition in the inhibition elisa , failed to detect any specific fluorescence indicative of true positives . the fact that the predictive value of a negative test is very high in low prevalence populations , even with tests of modest performance , further supports the conclusion that infection with n. caninum is unlikely . selection bias in the hpa sera collection is unlikely because of the provision of free access to healthcare for all by the national health service . moreover , a convenience collection has been shown to be comparable to a randomized sample in previous studies of population immunity ( 8) . the collection may therefore be considered to approximate the general population of england in terms of its exposure to n. caninum . in contrast , the phls farm cohort may be regarded as a relatively high - risk group for n. caninum infection since those persons would be predicted to have greater than normal exposure to bovine placentas , fetal membranes , and fluids that are potentially infected , or to environments contaminated by feces of dogs , which have access to tissues from potentially infected cattle . analyses to detect parasites or parasite dna have shown no definitive evidence of human infection with n. caninum ( 1 ) , but seropositivity has been reported in 3 studies ( 911 ) although not in 2 other european surveys ( 12,13 ) . of the 3 reports describing seropositivity , 2 ( 10,11 ) noted a high correlation with seropositivity to t. gondii , and , in all , antibody titers versus n. caninum antigen were low ( at < 1:100 dilution ) . the interpretation of serologic results is difficult , and this study s approach , in which large numbers of samples were quantitatively assayed to give a frequency distribution , may provide a helpful means of addressing this uncertainty . no evidence of human exposure to n. caninum was found in a high - risk population in england sampled in 1995 and in a sample of the general population of england collected in 2000 . however , given global variation in infection prevalence in cattle and possible regional differences in the incidence of oocyst shedding by dogs , there remains a need worldwide to remain vigilant to the possibility of human infection .
retrospective testing of 3,232 serum samples from the general population and 518 serum samples from a high - risk group showed no evidence of human exposure to neospora caninum in england . results were obtained by using immunofluorescence antibody testing and elisa to analyze frequency distribution .
You are an expert at summarizing long articles. Proceed to summarize the following text: a recent paper by martinez et al . in this journal reported the genome sequencing and analysis of five additional dermatophyte species , bringing the total number to seven ( 1 ) . in this commentary , we will situate this report in the context of current dermatophyte genomics and speculate on the future of the field based on the advances made in aspergillus genomics after the first three aspergillus genomes were sequenced in 2005 ( 24 ) . dermatophytes are a uniquely pathogenic group of fungi that cause most common fungal infections globally ( 5 ) . dermatophytic fungi are contained within three genera , trichophyton , epidermophyton , and microsporum . in the united states alone , millions of individuals seek treatment for dermatophyte infections annually , translating into an economic burden estimated at $ 400 million per year ( 6 ) . moreover , large - scale epidemics have been reported in american troops in conflicts in europe and an urban childcare center outbreak ( 7 , 8) . the knowledge surrounding the mode by which these pathogens cause disease is insufficient , perhaps due to lack of research utilizing modern molecular tools . due to this deficiency genetic tools have been underutilized in the characterization of these fungi , resulting in a lack of sequenced dermatophyte genomes and their pathogenicity ( 9 ) . as noted , seven whole - genome sequences of dermatophyte species have now been generated ( see the broad institute s dermatophyte comparative database at http://www.broadinstitute.org/annotation/genome/dermatophyte-comparative/multihome.html ) : the nuclear genome and mitochondrial sequences of microsporum canis , microsporum gypseum , trichophyton equinum , trichophyton rubrum , and trichophyton tonsurans ( 1 ) , as well as the availability of arthroderma benhamiae and trichophyton verrucosum genome sequences ( 10 ) . in their comparative study , martinez et al . ( 1 ) report that the sequenced dermatophytes are enriched relative to other human - associated fungi with four gene families that contribute to their ability to cause disease , an observation that mirrors the original analysis of the first two dermatophyte genomes ( 10 ) . these include ( i ) proteases , secreted to degrade skin , that reportedly act as virulence factors ; ( ii ) kinases , including pseudokinases , that are involved in signaling necessary for adapting to the skin niche ; ( iii ) secondary metabolites , compounds that act as toxins , immune system modulators , or signals in the interactions between fungus and host ; and ( iv ) a class of proteins ( lysm ) that appear to bind and mask cell wall components and carbohydrates , thus avoiding the host s immune response to the fungi . overall , these genome sequence identifications are important for revealing genome components that have the potential to further our understanding of the pathogenicity of dermatophytes . the availability of these sequence and analysis data will provide researchers large amounts of useful information that will provide power to studies aimed to decipher and interpret the molecular basis of host colonization , invasion , and specialization . the observations about the dermatophyte genomes are reminiscent of the observations made on the first three aspergillus genomes that were sequenced and analyzed . this is not surprising given that all dermatophytes and aspergilli belong to the same phylum , ascomycota . characterization and analysis of many virulence - associated traits in aspergillus species ( 1 ) may be useful in the search for such traits in dermatophyte genomes . additionally , aspergillus pathogens have been the subject of medically important research , targeting genes associated with replication cycles and secreted enzymes involved in secondary metabolite production . the genome sequences of aspergillus fumigatus , aspergillus nidulans , and aspergillus oryzae were reported in back - to - back nature papers in 2005 ( 24 ) . shortly after that publication event , the sequence of aspergillus flavus was completed ( 11 ) . a. fumigatus and a. flavus cause invasive aspergillosis in immunocompromised patients , an ability that positions them as the more important fungal pathogens in this group . all of these fungi but a. oryzae are environmental saprophytes whose niche is decaying plant material . a. oryzae , whose genome sequence revealed it to be essentially a derivative of ancestral a. flavus , has experienced centuries of human cultivation as a key ingredient in the production of sake , miso , soy sauce , and other japanese foods . at the time of the genome sequence publications , all but a. nidulans were presumed to be asexual . as was the case for dermatophytes , the genome analysis of these aspergilli revealed several striking features , including a surprising abundance of secondary metabolite biosynthetic gene clusters , a full set of sexual cycle genes even in the presumed asexual strains , and an abundance of secreted degradative enzymes . the aspergillus genomes inspired a burst of studies that leveraged these genome sequences , as overviewed in fig . 1 ( 1220 ) . subsequent post - genome sequence studies have revealed the identity of numerous products of the secondary - metabolite biosynthetic clusters and the roles of some of them in a. fumigatus and a. flavus virulence ; have identified conditions for in vitro sexual cycles in a. fumigatus and a. flavus , which has led to genetic analysis studies of these organisms that are now under way ; and have supported studies of the roles of many of the secreted proteases and other degradative enzymes in virulence . in addition , multiple strains of a. fumigatus and a. flavus have been sequenced , with the total of a. fumigatus sequences completed or under way approaching 100 ( http://gsc.jcvi.org/projects/gsc/a_fumigatus/index.php ) . the timeline highlights some of the numerous critical studies since the publication of the first three aspergillus genome sequences in 2005 . the cited papers should be taken as representative , as no rigorous prioritization was imposed in selecting papers to highlight in the timeline . sexual reproduction has been suggested as a means to revamp the virulence of fungi via meiotic recombination , which increases the population diversity , and via mating on the human host . these may be associated with antifungal resistance or the rate of pathogenicity of dermatophytes ( 21 ) . like the story of sex in a. fumigatus , some dermatophytes , which were once assumed to be asexual , based on these studies , prediction of unexposed sexual cycles can be assumed from the dermatophytes containing functional sex genes . recently , identification of the mating type locus ( mat ) of five dermatophytes ( m. canis , m. gypseum , t. equinum , t. rubrum , and t. tonsurans ) with comparable virulence were reported using bioinformatic tools ( 23 ) . furthermore , successful mating of t. rubrum with arthroderma simii suggests that these species have the benefits of sex , including cross - species sexual recombination and adaptation , that may outweigh the efficiencies of an asexual clonal expansion ( 24 ) . in the aspergillus species , the apn2 and sla2 genes , encoding a dna lyase and cytoskeleton protein , flank the mat loci ( 25 ) . however , those mat genes for dermatophytes are essentially identical and linked on one side of the mat locus ( 23 ) . the discovery and characterization of the mat locus of dermatophytes allows further studies in the pathogenesis to be explored . the importance of these proteins in avoiding detection by the host immune system is supported by the observation that during dermatophyte infection , defective or absent cell - mediated immunity predisposes the host to chronic or recurrent dermatophyte infection ( 26 ) . previously , expression of hydrophobin has been demonstrated to inhibit immune recognition in a. fumigatus ( 27 ) . dermatophytes a. benhamiae and t. verrucosum , both shown to activate human inflammatory infections , also display moderate expression of a surface hydrophobin gene , suggesting a possible role in immune response functions ( 10 ) . discovery of the abundance of secondary - metabolite biosynthetic clusters in the aspergillus genomes has led to the identification of the products of many of these clusters and the roles of some of them in virulence . a similar abundance of these clusters has now been noted in the reported dermatophyte genome sequences . for example , melanin , which is an important virulence determinant in aspergillus ( 28 ) , was also isolated from dermatophytes ( m. canis , m. gypseum , t. equinum , t. rubrum , and t. tonsurans ) in vitro and during infection , suggesting a similar role in aspergillus and dermatophyte pathogenesis ( 29 ) . moreover , t. rubrum produces xanthomegnin , a toxin produced by aspergillus in culture and in the human host ( 30 ) . transcriptome analysis revealed differential expression of secondary - metabolite genes during dermatophyte and aspergillus infections , underscoring their importance in the colonization of tissues and potentially in the manipulation of the host inflammatory response ( 30 ) . future studies will undoubtedly leverage the genome sequences of these clusters in dermatophytes to identify their secondary - metabolite products and their potential specific roles in virulence . given the recent major progress in the development of broad - scale transcriptional and genome sequence - dependent analyses of dermatophytes ( 10 , 3032 ) and a selection of functionally characterized genes ( 33 , 34 ) , full genome sequences will fulfill a critical urgency in the need to develop molecular genetic techniques to study these pathogens . molecular studies of dermatophyte genomics and pathogenicity have been undertaken in spite of the limited number of sequenced genomes . for example , vermout and colleagues used rna silencing as a potential functional genomics tool in m. canis to identify two proteases , sub3 and dppiv , coding for subtilisin and dipeptidyl peptidase , respectively ( 35 ) . previous studies have also demonstrated the association of increased keratinase with increased disease symptoms in m. canis ( 36 ) . several studies have used proteomics to characterize secreted and conidial proteins in t. rubrum , a. benhamiae , and m. canis ( 10 , 37 , 38 ) , but these have been limited in number and applicability by the lack of genome sequence . now that they can be coupled to genome sequences , these and other omics methods , such as metabolomics , glycomics , and lipidomics , will be more powerful , and accordingly , will strengthen the understanding and characterization of dermatophyte pathogenesis . it is clear that the availability of additional dermatophyte genomes will accelerate and enhance molecular studies of these pathogenic fungi . it is therefore most appropriate to celebrate the publication of these new dermatophyte genomes and to note this event as a consequential milestone in the efforts to manage the terrible diseases caused by this group of fungi . the aspergilli and the dermatophytes are closely related , and the new dermatophyte genome sequences reveal features similar to those in the aspergilli . this observation suggests commonality in how these fungi survive and thrive in a mammalian host . specific features of the dermatophytes and aspergilli diseases such as invasiveness , fatality , and organ involvement have resulted in research communities with disproportionate funding support that favored more rapid advancement in the aspergilli than in dermatophytes . another important factor that favored the aspergilli was the strength of a. nidulans as a model organism and the mature community that had developed around this model prior to the genome sequence publications . however , studies of these two groups of fungi have been , and will continue to be , synergistic , with each community taking lessons from the other . we project that the rate of progress in dermatophyte genomic research will accelerate now in much the same way aspergillus research accelerated following the publication of the aspergillus genomes in 2005 and 2006 .
abstractdermatophytes are a uniquely pathogenic group of fungi that cause most common fungal infections globally . the major cause of athlete s foot is trichophyton rubrum , a pathogen of human skin . a recent paper in this journal reported the sequencing and analysis of five additional genome sequences , including that of trichophyton rubrum . these five join the existing two additional genome sequences to bring the total to seven dermatophyte genome sequences , a notable milestone in the study of these fungi . these additional genomes set the stage for future genome - supported studies on the biology , pathogenicity , and host specificity of this important group of pathogens . to predict how this future might play out , we review the history of aspergillus genomics since the initial publication of the first three aspergillus genome sequences in 2005 , an event that stimulated important studies of the pathogenic aspergillus species . from these 7 years of aspergillus history , we offer some speculation on the future of dermatophyte studies supported by the genome sequences given the similarities , differences , and relative levels of support for studies in these two groups of fungi and the diseases they cause .
You are an expert at summarizing long articles. Proceed to summarize the following text: composting is the simplest traditional animal manure management technology that depends on the degradation of organic matter by the microbial community within manure itself ( bernal et al . , 2009 ) . easily degradable organic matter would be utilized as the energy source , and co2 , nh3 and moisture would be emitted and would generate large amounts of heat ; the temperature inside compost piles is about 70c . the mass of the pile decreases significantly , and the process also reduces odorous compounds and pathogens , while killing weed seeds . because the mature product can be reused as organic fertilizer , composting is a very important technology from the viewpoint of the circulation of resources or environmental protection . because the organic or inorganic state of nitrogen contained within the compost is an important nutrient for crops , the available amount of nitrogen content in composted material is a precious component . through the composting process , the organic nitrogen contained within initial fresh manure is degraded into ammonium by a wide variety of microorganisms including bacteria and fungi . part of this nitrogen is lost as nh3 by volatilization , or through conversion into gases content such as n2o or n2 through the nitrification / denitrification process ( fig . 1 ) . the range of nitrogen loss can vary between 19% and 77% , which mainly occur through nh3 volatilization and n2 emission ( martins and dewes , 1992 ; mahimairaja et al . , 1995 ; eghball et al . , 1997 ; tiquia and tam , 2000 ; tiquia et al . , 2002 ) . in addition , 0.29.9% of initial nitrogen content can be emitted as n2o , the intermediate of denitrification or byproducts of nitrification ( kuroda et al . , 1996 ; hao et al . , 2001 ; 2004 ; fukumoto et al . , 2003 ; el kader et al . the loss of nitrogen during animal manure composting processes is affected by various parameters , such as the animal species , diet , bulking agents , moisture content , turning frequency , carbon / nitrogen ratio and initial nitrogen content . nitrous oxide is an important greenhouse gas with strong global warming potential ( 300 times as that of co2 ( ipcc , 2001 ) . moreover , because n2o is greatly responsible for ozone depletion , reduction of its emission would be important for environmental protection ( ravishankara et al . , 2009 ) . therefore , an important issue in the study of composting is the nitrifier / denitrifier microbial community , which plays a significant role in nitrogen conversion within the composting pile . in this review article , we deal with recent research activities that focus on the nitrifier / denitrifier microbial community in composting while referring to similar studies in other environments . most of them focus on the bacteria mainly responsible for the degradation of organic matter . in order to identify microbes present in the compost process , besides the classical isolation technique , new approaches based on cultureindependent techniques , such as the extraction of dna from the compost and amplification of 16s rrna gene by pcr , followed by dna sequencing are commonly used ( muyzer et al . , 1993 ) . the approach based on dna sequencing provides relevant information on microbes that are difficult to culture . it has been reported that some bacillus species are important in the composting pile in the thermophilic stage , when active degradation of organic compounds occurs ( blanc et al . ; peters et al . , 2000 ; dees and ghiorse , 2001 ; zhang et al . , 2002 ; , 2003 ; iida et al . , 2005 ; kim et al . , 2006 ; wang et al . , 2007 ; yamamoto et al . , these bacillus can grow and degrade organic compounds under thermophilic conditions up to 65c , and thermus species are dominant instead of bacillus species above 70c ( beffa et al . , 1996 ) . while this is relevant , it should be noted that the bacterial community structure changes dramatically even in the maturing phase , when active degradation of organic compounds has almost ended . in the maturing phase , mesophilic proteobacteria or actinobacteria are known to be dominant ( danon et al . , 2008 ) , and , the temperature in the core zone of the pile reaches 6070c , and there are temperature gradient effects within the pile ( fernandes et al . , 1994 ) . in addition , there is an oxygen gradient and anoxic conditions deep inside the piles ( hao et al . , 2001 ) , especially in passively aerated composting systems . in these various complicated environments , bacterial communities differ significantly between the surface and the core zone ( maeda et al . , 2010a ) . in the dairy cattle manure composting process , nitrite and nitrate accumulate on the surface layer of the pile even in the initial stage of the process , when there are still easily degradable organic compounds ( maeda et al . , 16s rrnadependent bacterial community analysis suggests that some proteobacteria or bacteroidetes are dominant , which is significantly different from the case in thermophilic core zones . some part of these bacterial species are thought to contribute to the nitrification or denitrification that actively occurs in the surface layer . although dees and ghiorse ( 2001 ) reported that they failed to detect archaea in the compost piles , while they found many fungal species in the compost samples whose temperatures did not exceed 50c . in this regard , anastasi and colleagues ( 2005 ) reported the isolation of 194 fungal species , the acremonium , aspergillus , cladosporium , malbranchea , penicillium , pseudallescheria and thermomyces species from compost . in another study , hultman and colleagues ( 2009 ) reported that fungal biomass can represent between 6.3% and 38.5% of total biomass in municipal waste compost based on phospholipid fatty acid analysis . they also found that the fungal community suffers dramatic changes during the composting process , as does the bacterial community , and that a fungal community succession differed between a fullscale composting facility and a laboratoryscale small reactor . studies are needed on the function of the fungal community in the degradation of organic matter in the huminification process , or potential interaction with the bacterial community and its contribution to the nitrification / denitrification pathway . nitrification is known to be carried out by bacteria , archaea and fungi ( de boer and kowalchuk , 2001 ; leininger et al . , 2006 ; laughlin et al . , 2008 ) . in the bacterial process , nitrification consists of two steps , ammonia oxidation and nitrite oxidation , and each of these reactions is performed by an individual microbial group : ammoniaoxidizing bacteria ( aob ) ( kowalchuk and stephen , 2001 ) and nitriteoxidizing bacteria ( nob ) respectively . betaproteobacterial aob or thaumarchaeota ammoniaoxidizing archaea ( aoa ) are considered important in ammonia oxidation ( brochierarmanet et al . , 2008 ) , and the major nob in the environment are alphaproteobacterial nitrobacter or nitrospira . nitrifiers grow slowly under laboratory conditions , and their cultivation or isolation is very timeconsuming . in order to speed the tracking of these microbes , a molecular biology approach using primers specific to the 16s rrna genes and the ammonia monooxygenase gene of betaproteobacterial aob have been developed and used to track nitrifiers in the environment ( innerebner et al . , 2006 ; junier et al . , temporal nitrite accumulation in the middle stage and high accumulation of nitrate in the mature phase were observed ( he et al . , 2000 ; 2001 ; fukumoto et al . , 2006 ; fukumoto and inubushi , 2009 ; maeda et al . , 2010c ) , and it is evident that nitrification occurs in the compost pile . some studies have detected sequences similar to known aob species nitrosomonas europaeaeutropha or nitrosospira in the composting process ( kowalchuk et al . , 1999 ; jarvis et al . , 2009 ; maeda et al . jarvis and colleagues ( 2009 ) also detected nitrosomonas in the theromophilic stage and nitrosospira in the maturation phase of household waste composting , while maeda and colleagues ( 2010b ) detected nitrosomonas throughout the process , especially from the surface layer of a cattle manure composting pile . some previous studies also detected nitrosomonas from landfill cover , an organicrich environment similar to the composting process ( mertoglu et al . , 2006 ; zhu et al . , 2007 ) . betaproteobacterial aob are chemoautotrophic and generate energy from the hydroxylamine oxidation step , the atp produced is used to fix co2 as a carbon source . therefore , the presence of these aob indicates that these bacteria oxidize ammonia in the composting process . however , the extent of the contribution to net nitrification is yet unknown . to clarify the contribution of aob to ammonia oxidation in composting although aoa is known to be responsible to some extent for nitrification in environments containing less organic matter , such as soil , ocean or river sediment ( leininger et al . , 2006 ; santoro et al . , 2010 ) , there are some reports that aob 's contribution is much more important than aoa 's for actual nitrification in organicrich environments such as wastewater from treatment plants ( park et al . , 2006 ; another report shows that ammonia oxidation in zinccontaminated soil is restored not by aoa but by aob ( mertens et al . , 2009 ) . many heavy metals are included in livestock manure , especially in swine manure ( nicholson et al . , 1999 ; ko et al . , 2008 ) . although a previous report failed to detect the aoa in compost piles ( maeda et al . , 2010b ) , yamamoto et al . ( 2010 ) reported the existence of aoa from cattle manure compost in the later stage of the process . heterotrophic nitrification is a reaction in which heterotrophic bacteria oxidize ammonia or degrade organic matter to nitrate directly ( papen and von berg , 1998 ) . many bacterial species are known to undergo this reaction , and species such as paracoccus denitrificans or pseudomonas putida are known to possess amoa sequences distinct from those of autotrophic nitrifiers ( moir et al . , 1996 ; daum et al . , 1998 ) . although these bacteria have potential to contribute net nitrification in the compost , its actual contribution is not known at all . this reaction by heterotrophs has not been considered in depth because these species do not generate energy from this process , nor do they accumulate high concentrations of nitrite ; however , this futile reaction may be of relevance in an environmental setting . these heterotrophic nitrifiers assimilate more ammonium than chemoautotrophic aob , which leads to higher biomass , and they have been considered not useful for wastewater treatment systems ( podmirseg et al . , 2010 ) . efforts to unveil nitrification process include the development of new culture media for thermophilic nitrifiers in compost under heterotrophic conditions is ongoing ( shimaya and hashimoto , 2008 ) . in summary , it can be said that to understand the nitrogen cycle in the composting process , we need to learn more about the role of the heterotrophic nitrifiers in the process . on the other hand , there have not been many molecular ecological studies on nob . because nob has a diverse taxonomy , including nitrobacter ( proteobacteria ) , nitrosococcus mobilis ( proteobacteria ) , nitrospina gracilis ( proteobacteria ) and nitrospira ( nitrospira ) , it is difficult to detect all these strains by methods such as fish ( fluorescent in situ hybridization ) or pcr , which depend on 16s rrna sequences . few studies have focused on functioning gene sequences of nitrite oxidoreductase of nitrobacter ( poly et al . , 2008 ; wertz et al . , 2008 ) . even though these methods may be effective for understanding nitrification in the composting process , they have not been used for studies of nitrite oxidation yet , and nob in the composting process has not been characterized well . nitrite or nitrate generated by nitrifiers would usually be reduced by heterotrophic denitrifiers and emitted into the atmosphere as n2o or n2 . denitrification by bacteria has been well studied and the details of its molecular mechanisms have been characterized . the reaction consists of four reduction steps , namely , no3 no2 no n2o n2 . the genes nar , nir , nor and nos are coding the catalysing enzymes ( rudolf and kroneck , 2005 ; tavares et al . , 2006 ) . denitrifying bacteria are known to be phylogenetically diverse , with at least 50 genera ( zumft , 1997 ) . therefore , the study of denitrifiers that depend on 16s rrna gene sequences is very difficult , and functioning genes that code each enzyme catalysing each denitrification step are frequently used for studies of environmental denitrifiers ( sharma et al . , 2005 ; because of the relative abundance of information in public databases , nitrite reductase ( nirs and nirk ) or nitrous oxide reductase ( nosz ) have been used frequently . there are two types of nitrite reductase : nirs , cytochrome c nitrite reductase , which has haem iron in its active centre ( einsle et al . , 1999 ; 2000 ) , and nirk , a coppercontaining nitrite reductase ( murphy et al . , 1997 ; antonyuk et al . , 2005 ) . it is possible to distinguish these nitrite reductases in denitrifiers by using diethyldithiocarbamate ( ddtc ) , which chelates copper of the nirk denitrifier and prevents the process . dgge primers targeting nitrite reductase gene sequences have been developed ( throback et al . , it is frequently discussed which types of nitrite reducers would be dominant in the environment . for example , one report shows that nirs denitrifiers are dominant in subtropical macrotidal estuaries ( abell et al . , 2009 ) . because the horizonal transfer of denitrifying genes may occur within the environment , the incidence of nirk or nirs does heylen and colleagues ( 2006a ) concluded that nir genes may not be suitable to evaluate microbial diversity of denitrifiers in the environment . thus , interpretation of biodiversity based on nir sequence analysis need to be interpreted with care . nitric oxide , produced by the reduction of nitrite , is known to be toxic to microorganisms , and they need to metabolize it to protect themselves . three kinds of nor have been reported , namely cnor , qnor and qcunor ( tavares et al . , 2006 ) . the cytochrome cdependent nitric oxide reductase ( cnor ) of p. denitrificans has been studied well . it is a component of cytochrome bc complex with two nonhaem irons in its active centre . braker and tiedje ( 2003 ) were the first to study denitrifying communities using norb as a functional marker , and others have used it for studies on environmental samples ( dandie et al . , 2007 ) or isolates ( heylen et al . , nitrous oxide reductase is the terminal oxidoreductase of denitrification that transforms n2o to n2 ( brown et al . , 2000 ) . because this multicoppercontaining enzyme prevents the accumulation of a potent greenhouse gas , it plays an important role in the nitrogen cycle ( zumft , 2005 ) . nitrous oxide reductase is most sensitive to molecular oxygen among the enzymes involved in denitrification , and its function is inhibited under aerobic conditions . this enzyme can also be inhibited by c2h2 easily ( yoshinari and knowles , 1976 ) and is frequently used for the study of the denitrification potential of environmental samples ( teissier and torre , 2002 ) . moreover , the nosz gene that codes this enzyme is used as a biomarker in molecular ecological studies ( scala and kerkhof , 1999 ; stres et al . , 2004 ) . some bacterial denitrifiers and fungi are known not to possess nitrous oxide reductase ( takaya , 2009 ) . although n2o reduction is thermodynamically favourable and n2o is suitable for an electron acceptor , some denitrifiers produce n2o as the final product of the denitrification process . this might be explained by the fact that nitrous oxide is not toxic to microorganisms , whereas no is toxic to bacterial cells . the lack of n2o reduction makes 20% difference to the bioenergetics of the bacterium ( richardson et al . , 2009 ) . a study about denitrifier communities in the composting process revealed an initial variation of nirk diversity and stability after that ( maeda et al . , 2010a ) . hallin and colleagues ( 2006 ) also reported that the addition of methanol or ethanol to activated sludge significantly affected the diversity of nirs but not that of nirk . on the other hand , the addition of mature compost that contains no2 or no3n did not affect nirk diversity but significantly affected nosz diversity , suggesting that denitrifiers possessing the nosz gene in the compost would be more sensitive to environmental conditions . it is necessary to isolate the major denitrifier revealed by molecular methods in order to understand the actual denitrification occurring in the environment . in a denitrifier community study of rice paddy soil , ashida and colleagues ( 2010 ) successfully isolated a major denitrifier through the enhancement of denitrification activity with succinate amendment and molecular methods such as the 16s rrna gene clone library approach ( ishii et al . , 2009 ) or the stable isotope probing approach ( saito et al . , 2008 moreover , ishii and colleagues ( 2011 ) proved that denitrifiers with different 16s rrna gene phylogeny possess same nirs or nirk gene in the same environment ( fig . 2 ) . their data show previously unknown complex relationship between 16s rrna gene and functional gene possession . to understand the denitrifier community completely , it is necessary to combine independent approaches such as molecular and conventional cultivation approaches . the molecular methods used to characterize the unknown and uncultivated denitrifier communities , and the subsequent singlecell isolation strategy would be effective for the denitrifiers that are truly functioning for actual denitrification in the environment ( ishii et al . , 2010 ) . the relationship of 16s rrna gene and functional denitrifier genes ( nirs , nirk ) possession of denitrifiers isolated from rice paddy soil ( redrawn from ishii et al . , 2011 ) . isolated strains were written in bold , and the possession relationship was connected by dotted lines . the relationship between denitrifying gene diversity or abundance and potential denitrification activity in soil has been well studied . potential denitrification activity , n2o/(n2o + n2 ) and the denitrifier community would be affected by ph ( palmer et al . , on the other hand , hallin and colleagues ( 2009 ) reported that denitrification activity did not correlate with denitrifier gene composition , but did correlate with the size of the total bacterial community or nosz abundance . another study reports that the nosz ratio to total bacterial community is much more important than denitrifying gene abundance for potential n2o production ( philippot et al . , 2009 ) . although much effort has been made in these environmental studies , it is still difficult to explain denitrification and nitrous oxide production by denitrifying gene abundance or diversity . moreover , a previous paper reported that aoa possesses a novel nirk sequence ( bartossek et al . , 2010 ) , which had not been covered by previous denitrifier studies , and much effort should be made to learn about this unknown archaean denitrification . it is also known that some autotrophic nitrifiers have the ability to denitrify ( wrage et al . , 2001 ; these autotrophic nitrifiers possess nirktype nitrite reductase with distinct dna sequences from those of heterotrophic denitrifiers . it is not well understood yet how these autotrophic nitrifiers acquired the nirk gene , which might have occurred by horizontal gene transfer ; and how they became tolerant to nitrite produced by themselves ( casciotti and ward , 2001 ) . because nitrifiers produce nitrite , they have many advantages for the utilization of nitrite as the substrate for denitrification . therefore , nitrifier denitrification may contribute much more than heterotrophic denitrifiers , but it is difficult to distinguish these pathways with the current analytic techniques . as stated above , nitrification would be performed by aob , aoa , heterotrophic nitrifiers or fungi , whereas denitrification would be performed by heterotrophic denitrifiers , denitrifying fungi and autotrophic / heterotrophic nitrifiers . however , the relative contribution to net nitrification or denitrification of each group is not yet clear . studies that focus on nitrifying genes are only about aob or aoa , and those for denitrifying gene analysis are only about bacteria . the development of a tool for such study will be needed . stable isotope analysis of n2o is an alternative approach to studying its production processes because the relative abundance of stable isotopes is a function of their abundance in source materials and the isotope fractionation factor of each physical / chemical process . in particular , intramolecular n distribution within the n2o molecule ( site preference , sp ) has been found to depend only on enzymatic reaction processes and not on substrates ( toyoda and yoshida , 1999 ; yoshida and toyoda , 2000 ; toyoda et al . , 2005 ; sutka et al . , nitrous oxide , which originates from bacterial nitrification ( hydroxylamine oxidation ) and denitrification ( nitrite reduction ) , can be distinguished by using sp . however , sp can not distinguish between nitrifier denitrification and heterotrophic denitrification , and a recent study showed that fungal denitrification produces n2o with sp similar to that of bacterial nitrification ( sutka et al . in addition , isotope abundance is affected by nitrous oxide reduction ( ostrom et al . , 2007 ; jinuntuyanortman et al . , 2008 ) . although this analytical technique has some limitations as stated above , it would be a powerful tool by using all available isotopic data ( n and o isotope ratios and sp ) in a complementary style ( e.g. koba et al . , 2009 ) or by combining other analytical approaches , such as a wide range of molecular methods . isotopomer analysis of n2o directly collected from a composting pile by the dynamic chamber method ( osada and fukumoto , 2001 ) revealed that bacterial denitrification is the most important and responsible nitrous oxide production pathway ( maeda et al . , 2010b ) . this study relied on the isotopic characteristics of n2o produced by isolates not from compost but from other environments ( such as soil ) . therefore , in future studies , we need to isolate the major nitrifier , denitrifier , denitrifying fungi and isotope signature of their producing n2o . nitrification is known to be carried out by bacteria , archaea and fungi ( de boer and kowalchuk , 2001 ; leininger et al . , 2006 ; laughlin et al . , 2008 ) . in the bacterial process , nitrification consists of two steps , ammonia oxidation and nitrite oxidation , and each of these reactions is performed by an individual microbial group : ammoniaoxidizing bacteria ( aob ) ( kowalchuk and stephen , 2001 ) and nitriteoxidizing bacteria ( nob ) respectively . betaproteobacterial aob or thaumarchaeota ammoniaoxidizing archaea ( aoa ) are considered important in ammonia oxidation ( brochierarmanet et al . , 2008 ) , and the major nob in the environment are alphaproteobacterial nitrobacter or nitrospira . nitrifiers grow slowly under laboratory conditions , and their cultivation or isolation is very timeconsuming . in order to speed the tracking of these microbes , a molecular biology approach using primers specific to the 16s rrna genes and the ammonia monooxygenase gene of betaproteobacterial aob have been developed and used to track nitrifiers in the environment ( innerebner et al . , 2006 ; junier et al . , temporal nitrite accumulation in the middle stage and high accumulation of nitrate in the mature phase were observed ( he et al . , 2000 ; 2001 ; fukumoto et al . , 2006 ; fukumoto and inubushi , 2009 ; maeda et al . , 2010c ) , and it is evident that nitrification occurs in the compost pile . some studies have detected sequences similar to known aob species nitrosomonas europaeaeutropha or nitrosospira in the composting process ( kowalchuk et al . , 1999 ; jarvis et al . , 2009 ; maeda et al . jarvis and colleagues ( 2009 ) also detected nitrosomonas in the theromophilic stage and nitrosospira in the maturation phase of household waste composting , while maeda and colleagues ( 2010b ) detected nitrosomonas throughout the process , especially from the surface layer of a cattle manure composting pile . some previous studies also detected nitrosomonas from landfill cover , an organicrich environment similar to the composting process ( mertoglu et al . , 2006 ; zhu et al . , 2007 ) . betaproteobacterial aob are chemoautotrophic and generate energy from the hydroxylamine oxidation step , the atp produced is used to fix co2 as a carbon source . therefore , the presence of these aob indicates that these bacteria oxidize ammonia in the composting process . however , the extent of the contribution to net nitrification is yet unknown . to clarify the contribution of aob to ammonia oxidation in composting although aoa is known to be responsible to some extent for nitrification in environments containing less organic matter , such as soil , ocean or river sediment ( leininger et al . , 2006 ; santoro et al . , 2010 ) , there are some reports that aob 's contribution is much more important than aoa 's for actual nitrification in organicrich environments such as wastewater from treatment plants ( park et al . , 2006 ; another report shows that ammonia oxidation in zinccontaminated soil is restored not by aoa but by aob ( mertens et al . , 2009 ) . many heavy metals are included in livestock manure , especially in swine manure ( nicholson et al . , 1999 ; ko et al . , 2008 ) . although a previous report failed to detect the aoa in compost piles ( maeda et al . , 2010b ) , yamamoto et al . ( 2010 ) reported the existence of aoa from cattle manure compost in the later stage of the process . heterotrophic nitrification is a reaction in which heterotrophic bacteria oxidize ammonia or degrade organic matter to nitrate directly ( papen and von berg , 1998 ) . many bacterial species are known to undergo this reaction , and species such as paracoccus denitrificans or pseudomonas putida are known to possess amoa sequences distinct from those of autotrophic nitrifiers ( moir et al . , 1996 ; daum et al . , 1998 ) . although these bacteria have potential to contribute net nitrification in the compost , its actual contribution is not known at all . this reaction by heterotrophs has not been considered in depth because these species do not generate energy from this process , nor do they accumulate high concentrations of nitrite ; however , this futile reaction may be of relevance in an environmental setting . these heterotrophic nitrifiers assimilate more ammonium than chemoautotrophic aob , which leads to higher biomass , and they have been considered not useful for wastewater treatment systems ( podmirseg et al . , 2010 ) . efforts to unveil nitrification process include the development of new culture media for thermophilic nitrifiers in compost under heterotrophic conditions is ongoing ( shimaya and hashimoto , 2008 ) . in summary , it can be said that to understand the nitrogen cycle in the composting process , we need to learn more about the role of the heterotrophic nitrifiers in the process . on the other hand , there have not been many molecular ecological studies on nob . because nob has a diverse taxonomy , including nitrobacter ( proteobacteria ) , nitrosococcus mobilis ( proteobacteria ) , nitrospina gracilis ( proteobacteria ) and nitrospira ( nitrospira ) , it is difficult to detect all these strains by methods such as fish ( fluorescent in situ hybridization ) or pcr , which depend on 16s rrna sequences . few studies have focused on functioning gene sequences of nitrite oxidoreductase of nitrobacter ( poly et al . , 2008 ; wertz et al . , 2008 ) . even though these methods may be effective for understanding nitrification in the composting process , they have not been used for studies of nitrite oxidation yet , and nob in the composting process has not been characterized well . nitrite or nitrate generated by nitrifiers would usually be reduced by heterotrophic denitrifiers and emitted into the atmosphere as n2o or n2 . denitrification by bacteria has been well studied and the details of its molecular mechanisms have been characterized . the reaction consists of four reduction steps , namely , no3 no2 no n2o n2 . the genes nar , nir , nor and nos are coding the catalysing enzymes ( rudolf and kroneck , 2005 ; tavares et al . , 2006 ) . denitrifying bacteria are known to be phylogenetically diverse , with at least 50 genera ( zumft , 1997 ) . therefore , the study of denitrifiers that depend on 16s rrna gene sequences is very difficult , and functioning genes that code each enzyme catalysing each denitrification step are frequently used for studies of environmental denitrifiers ( sharma et al . , 2005 ; because of the relative abundance of information in public databases , nitrite reductase ( nirs and nirk ) or nitrous oxide reductase ( nosz ) have been used frequently . there are two types of nitrite reductase : nirs , cytochrome c nitrite reductase , which has haem iron in its active centre ( einsle et al . , 1999 ; 2000 ) , and nirk , a coppercontaining nitrite reductase ( murphy et al . , 1997 ; antonyuk et al . , 2005 ) . it is possible to distinguish these nitrite reductases in denitrifiers by using diethyldithiocarbamate ( ddtc ) , which chelates copper of the nirk denitrifier and prevents the process . dgge primers targeting nitrite reductase gene sequences have been developed ( throback et al . , it is frequently discussed which types of nitrite reducers would be dominant in the environment . for example , one report shows that nirs denitrifiers are dominant in subtropical macrotidal estuaries ( abell et al . , 2009 ) . because the horizonal transfer of denitrifying genes may occur within the environment , the incidence of nirk or nirs does heylen and colleagues ( 2006a ) concluded that nir genes may not be suitable to evaluate microbial diversity of denitrifiers in the environment . thus , interpretation of biodiversity based on nir sequence analysis need to be interpreted with care . . nitric oxide , produced by the reduction of nitrite , is known to be toxic to microorganisms , and they need to metabolize it to protect themselves . three kinds of nor have been reported , namely cnor , qnor and qcunor ( tavares et al . , 2006 ) . the cytochrome cdependent nitric oxide reductase ( cnor ) of p. denitrificans has been studied well . it is a component of cytochrome bc complex with two nonhaem irons in its active centre . braker and tiedje ( 2003 ) were the first to study denitrifying communities using norb as a functional marker , and others have used it for studies on environmental samples ( dandie et al . , 2007 ) or isolates ( heylen et al . , nitrous oxide reductase is the terminal oxidoreductase of denitrification that transforms n2o to n2 ( brown et al . , 2000 ) . because this multicoppercontaining enzyme prevents the accumulation of a potent greenhouse gas , it plays an important role in the nitrogen cycle ( zumft , 2005 ) . nitrous oxide reductase is most sensitive to molecular oxygen among the enzymes involved in denitrification , and its function is inhibited under aerobic conditions . this enzyme can also be inhibited by c2h2 easily ( yoshinari and knowles , 1976 ) and is frequently used for the study of the denitrification potential of environmental samples ( teissier and torre , 2002 ) . moreover , the nosz gene that codes this enzyme is used as a biomarker in molecular ecological studies ( scala and kerkhof , 1999 ; stres et al . , some bacterial denitrifiers and fungi are known not to possess nitrous oxide reductase ( takaya , 2009 ) . although n2o reduction is thermodynamically favourable and n2o is suitable for an electron acceptor , some denitrifiers produce n2o as the final product of the denitrification process . this might be explained by the fact that nitrous oxide is not toxic to microorganisms , whereas no is toxic to bacterial cells . the lack of n2o reduction makes 20% difference to the bioenergetics of the bacterium ( richardson et al . , 2009 ) . a study about denitrifier communities in the composting process revealed an initial variation of nirk diversity and stability after that ( maeda et al . , 2010a ) . hallin and colleagues ( 2006 ) also reported that the addition of methanol or ethanol to activated sludge significantly affected the diversity of nirs but not that of nirk . on the other hand , the addition of mature compost that contains no2 or no3n did not affect nirk diversity but significantly affected nosz diversity , suggesting that denitrifiers possessing the nosz gene in the compost would be more sensitive to environmental conditions . it is necessary to isolate the major denitrifier revealed by molecular methods in order to understand the actual denitrification occurring in the environment . in a denitrifier community study of rice paddy soil , ashida and colleagues ( 2010 ) successfully isolated a major denitrifier through the enhancement of denitrification activity with succinate amendment and molecular methods such as the 16s rrna gene clone library approach ( ishii et al . , 2009 ) or the stable isotope probing approach ( saito et al . , 2008 moreover , ishii and colleagues ( 2011 ) proved that denitrifiers with different 16s rrna gene phylogeny possess same nirs or nirk gene in the same environment ( fig . 2 ) . their data show previously unknown complex relationship between 16s rrna gene and functional gene possession . to understand the denitrifier community completely , it is necessary to combine independent approaches such as molecular and conventional cultivation approaches . the molecular methods used to characterize the unknown and uncultivated denitrifier communities , and the subsequent singlecell isolation strategy would be effective for the denitrifiers that are truly functioning for actual denitrification in the environment ( ishii et al . , 2010 ) . the relationship of 16s rrna gene and functional denitrifier genes ( nirs , nirk ) possession of denitrifiers isolated from rice paddy soil ( redrawn from ishii et al . , 2011 ) . isolated strains were written in bold , and the possession relationship was connected by dotted lines . the relationship between denitrifying gene diversity or abundance and potential denitrification activity in soil has been well studied . potential denitrification activity , n2o/(n2o + n2 ) and the denitrifier community would be affected by ph ( palmer et al . , cuhel and colleagues ( 2010 ) reported that nirs diversity correlates with soil ph . on the other hand , hallin and colleagues ( 2009 ) reported that denitrification activity did not correlate with denitrifier gene composition , but did correlate with the size of the total bacterial community or nosz abundance . another study reports that the nosz ratio to total bacterial community is much more important than denitrifying gene abundance for potential n2o production ( philippot et al . , 2009 ) . although much effort has been made in these environmental studies , it is still difficult to explain denitrification and nitrous oxide production by denitrifying gene abundance or diversity . moreover , a previous paper reported that aoa possesses a novel nirk sequence ( bartossek et al . , 2010 ) , which had not been covered by previous denitrifier studies , and much effort should be made to learn about this unknown archaean denitrification . it is also known that some autotrophic nitrifiers have the ability to denitrify ( wrage et al . , 2001 ; wrage et al . , 2004 ; shaw et al . , these autotrophic nitrifiers possess nirktype nitrite reductase with distinct dna sequences from those of heterotrophic denitrifiers . it is not well understood yet how these autotrophic nitrifiers acquired the nirk gene , which might have occurred by horizontal gene transfer ; and how they became tolerant to nitrite produced by themselves ( casciotti and ward , 2001 ) . because nitrifiers produce nitrite , they have many advantages for the utilization of nitrite as the substrate for denitrification . therefore , nitrifier denitrification may contribute much more than heterotrophic denitrifiers , but it is difficult to distinguish these pathways with the current analytic techniques . as stated above , nitrification would be performed by aob , aoa , heterotrophic nitrifiers or fungi , whereas denitrification would be performed by heterotrophic denitrifiers , denitrifying fungi and autotrophic / heterotrophic nitrifiers . however , the relative contribution to net nitrification or denitrification of each group is not yet clear . studies that focus on nitrifying genes are only about aob or aoa , and those for denitrifying gene analysis are only about bacteria . the development of a tool for such study will be needed . stable isotope analysis of n2o is an alternative approach to studying its production processes because the relative abundance of stable isotopes is a function of their abundance in source materials and the isotope fractionation factor of each physical / chemical process . in particular , intramolecular n distribution within the n2o molecule ( site preference , sp ) has been found to depend only on enzymatic reaction processes and not on substrates ( toyoda and yoshida , 1999 ; yoshida and toyoda , 2000 ; toyoda et al . , 2005 ; sutka et al . , nitrous oxide , which originates from bacterial nitrification ( hydroxylamine oxidation ) and denitrification ( nitrite reduction ) , can be distinguished by using sp . however , sp can not distinguish between nitrifier denitrification and heterotrophic denitrification , and a recent study showed that fungal denitrification produces n2o with sp similar to that of bacterial nitrification ( sutka et al . , 2008 ) . in addition , isotope abundance is affected by nitrous oxide reduction ( ostrom et al . , 2007 ; jinuntuyanortman et al . , 2008 ) . although this analytical technique has some limitations as stated above , it would be a powerful tool by using all available isotopic data ( n and o isotope ratios and sp ) in a complementary style ( e.g. koba et al . , 2009 ) or by combining other analytical approaches , such as a wide range of molecular methods . isotopomer analysis of n2o directly collected from a composting pile by the dynamic chamber method ( osada and fukumoto , 2001 ) revealed that bacterial denitrification is the most important and responsible nitrous oxide production pathway ( maeda et al . , 2010b ) . this study relied on the isotopic characteristics of n2o produced by isolates not from compost but from other environments ( such as soil ) . therefore , in future studies , we need to isolate the major nitrifier , denitrifier , denitrifying fungi and isotope signature of their producing n2o . because of the ease with which it is managed , composting will continue to be a major technology for treating animal manure . however , analysing the techniques developed previously can not explain the nitrogen cycle and nitrous oxide emission yet . in the future , combining distinct approaches such as molecular methods , stable isotope analysis and classical isolation techniques will help us to understand the nitrogen cycle during the composting process in detail . the results should lead to the development of relevant mitigation strategies , which will include : identification of the main players in the nitrification denitrification process in the composting piles ; isolation of these key players ; and analysis of their physiological , biochemical and ecological properties . it would be also of interest to identify the nitrous oxide reducers and to study their function in the composting piles .
summarycomposting is the major technology in the treatment of animal manure and is a source of nitrous oxide , a greenhouse gas . although the microbiological processes of both nitrification and denitrification are involved in composting , the key players in these pathways have not been well identified . recent molecular microbiological methodologies have revealed the presence of dominant bacillus species in the degradation of organic material or betaproteobacterial ammoniaoxidizing bacteria on nitrification on the surface , and have also revealed the mechanism of nitrous oxide emission in this complicated process to some extent . some bacteria , archaea or fungi still would be considered potential key players , and the contribution of some pathways , such as nitrifier denitrification or heterotrophic nitrification , might be involved in composting . this review article discusses these potential microbial players in nitrification denitrification within the composting pile and highlights the relevant unknowns through recent activities that focus on the nitrogen cycle within the animal manure composting process .
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Proceed to summarize the following text: the processing of affect underpins human experience and serves as the foundation of human interactions in everyday life . this complex process involves the ability to perceive , understand , and respond appropriately to emotional cues . the accurate understanding of these emotional cues is crucial in the assessment of threat , in negotiating transactions , and in the formation of social bonds ( 1 - 4 ) . autism spectrum disorders ( asd ) is an umbrella term often used to describe a continuum of diagnoses that include autistic disorder , asperger s disorder , and pervasive developmental disorder - not otherwise specified ( pdd - nos ) . collectively , asd is characterized by deficits in communication , impairments in social interactions , and restricted and repetitive patterns of behavior ( 5 ) . one of the core deficits in individuals with asd is a deficit in emotion processing and communication that prevents them from achieving successful reciprocal social interactions . laushey and heflin ( 2000 ) have proposed that the most profound and defining issues for individuals with asd are those difficulties related to poor social functioning ( 6 ) . specific social deficits among individuals with asd often include difficulties in expressing emotions , understanding the emotions of others , and empathizing ( 7 - 11 ) , as well as the inability to interpret social cues , assess the formality of social events , and act accordingly ( 12 ) . the inability to understand the emotional expressions of others and respond appropriately hinders the development of meaningful interpersonal relationships . as a result , individuals with asd who lack the ability to successfully interpret emotional cues are at risk for social rejection . in fact , children with asd often report poorer quality friendships , including more loneliness , less companionship and less help from their friends ( 13 ) . thus , the ramifications of emotion processing difficulties frequently include both emotional and physical isolation . deficits in the ability to understand and interpret emotional stimuli among individuals with asd provides a unique and important opportunity for the study of emotion processing , both for clinical and typically developing populations . in fact , the processing of bodily expressions of emotion has been widely studied in individuals with asd ( 14 , 15 ) . however , musical emotion processing in asd has received very little research attention , despite the fact that individuals with asd seem to respond so positively to music ( 16 ) . the purpose of this review is to examine studies of emotion processing in typical development and in individuals with asd , with a focus on music perception and how it may affect the functioning of the mirror neuron system ( mns ) in individuals with autism . our ultimate aim is to evaluate the potential role of music in the study and treatment of socioemotional deficits in individuals with asd and highlight the need for studies of musical emotion processing , which may hold a promising key to the development of novel , music - based therapeutic interventions . perhaps the most fundamental level of social reciprocity is the ability to be in a relationship , which is founded upon the critical component of emotional attunement . the importance of affect exchange is evident from the earliest days of life , when social reciprocity begins with affect sharing and coordination with the caregiver . trevarthen and aitken ( 1994 ) have proposed that there is an intrinsic motive formation that acts as the foundation for the infant s adaptation to her / his environment . this pattern of neural activity works to direct the infant s brain emotionally as they self - regulate and try to understand the feelings of others ( 17 ) . these early exchanges of affect between caregivers and infants lay the foundation for the development of more complex types of communication later in life ( 18 ) . emotional attunement in typically developing children is documented as early as nine minutes of age , where infants preferentially attend to a face - like stimulus ( 19 ) , and demonstrate a preference for their mother s voice at birth ( 20 ) . infants as early as two to three months of age begin to discriminate between several different affective expressions shown by their parents ( 20 ) . at five months , infants are developing the ability to read affect in the faces of strangers and at six months are able to emotionally attune to their caregivers ( 21 ) . arguably , the foundation for emotional attunement and complex social interactions is dependent upon the emergence and development of joint attention . joint attention is defined as the ability to coordinate interest in external objects or events with other people ( 22 ) , and is thought to be an attempt to share the experience of objects and events with others ( 23 ) . among typically developing infants , the ability for joint attention emerges between 9 and 15 months ( 24 ) . this is when the infant learns to use gaze to direct the attention of others to objects of interest ( 25 ) . an inherent interest in other people appears to provide the motivation for some of the behavior that leads to learning to share and track gaze ( 26 - 29 ) . imitative behavior also becomes more prevalent , precise and object - directed at this time . for example , infants will typically imitate the ways in which adults manipulate objects ( 30 ) . studies have shown that beginning in early infancy , typically developing infants begin to use social referencing to make sense of confusing or threatening situations or persons , by looking at the face of their caregiver for information ( 31 , 32 ) . such behaviors that emerge around nine months and continue to develop into the third year of life increase the infants understanding of self and others as intentional agents , and are based in the gradual development of theory of mind ( 33 ) . theory of mind ( tom ) is the ability to understand the desires , intentions , and knowledge of another ( 34 ) . typically developing children generally show evidence of a tom around age four as they begin to understand the perspectives of other persons ( 35 ) however , based upon a study examining non - verbal looking time using the violation - of - expectation method , it has been argued that the emergence of a tom with an implicit understanding of beliefs may even begin to develop as early as 15 months ( 36 ) . in examining precursors to tom , sodian and thoermer ( 2008 ) show that children under the age of three have the ability to use their own and others epistemic states in communication when engaging in false - belief tasks ( 37 ) . as children continue to grow , their ability to read affective cues and socially reciprocate becomes more refined . the gradual acquisition of such knowledge allows one to be part of and function successfully in a given culture , by incorporating others perspectives into one s own mind through cultural learning ( 33 ) . compared to typically developing children , the developmental trajectory of affect attunement is very different among infants with asd . even in infancy , children with asd fail to show the typical interest in human faces , instead showing a preference for inanimate objects ( 38 - 41 ) . the automatic mirroring of affective gestures and expressions is also considerably weaker in infants with asd ( 42 - 44 ) . consequences related to these deficits are displayed during infancy , and extend throughout childhood development . children with asd tend to pay less attention to the negative emotions of others compared to typically developing children ( 45 - 47 ) . they also appear to have more negative affective exchanges ( 48 ) , and display less positive emotion to their social partner ( 49 , 50 ) when compared to typically developing peers . the lack of attention paid to emotional cues often causes a rift in social interactions as well as lowering the affective responsivity of children with asd . emotional reciprocity is fundamental to the development of social skills and successful social interactions . due to the combination of deficits exhibited by children with asd in this realm , deficits in joint attention among individuals with autism are well - documented ( 51 - 53 ) . mundy , sigman and kasari ( 1994 ) found that joint attention for children with asd was lower than developmentally delayed and typically developing children ( 24 ) . in a more recent study , naber and colleagues ( 2007 ) examined different types of joint attention , including basic , associated , and visual . at 24 months , children with asd showed significantly less overall joint attention correlated with characteristics of autism and developmental level , but at 42 months the associations disappeared . of the three types of joint attention , only joint visual attention was less at 42 months , suggesting that joint visual attention may be the distinct deficit of concern in the development of joint attention in children with asd ( 54 ) . in research on gaze behavior it has been found that , compared to typically developing children , young children with asd shift attention between an object and another object more often than between a person and object or between person and person ( 55 ) . furthermore , children with asd spend less time looking at people and more time looking at objects , compared to neurotypical children ( 55 ) . by contrast , the processing of cartoons with human figures does not show any difference between typically developing children and children with asd ( 56 ) . in a study by klin and colleagues ( 2002 ) , it was found that when looking at faces , adults with asd looked at the eyes only half the time of neurotypical adults , but twice as often at mouths and objects ( 57 ) . a recent neuroimaging study found that brain responses to non - social stimuli ( houses ) were modulated by attention , both in adults with asd and a matched control group , but only the control participants demonstrated attentional modulation of face - selective regions during viewing of social ( face ) stimuli ( 58 ) . thus , the participants with asd demonstrated a lack of attentional modulation for the social stimulus . taken together , these findings suggest that atypical gaze patterns in individuals with asd may be related to the reduced salience of social stimuli from the earliest days of life , a deficit that persists into adulthood . indeed , reduced visual attention to socially salient stimuli may underpin many of the social communication impairments seen in individuals with asd . while typically developing infants use social referencing to make sense of confusing or threatening situations ( 32 ) , children with asd are less likely to reference another person in these same instances ( 59 ) - they are unlikely to voluntarily share their experience with others , and avoid initiating triadic interactions , even when guided to do so ( 60 ) . however , these atypical social behaviors in children with asd have been suggested to be secondary to impairments in the ability to represent mental states of others , or tom ( 61 ) . normal intellectual and verbal abilities appear to lessen the impact of earlier impairment in tom , such that in structured situations , individuals with asd may actually demonstrate tom abilities ( 62 , 63 ) . tom has been studied in relation to several other aspects of asd , including how individuals with asd are less likely to utilize tom to spontaneously attribute meaning ( 64 ) , in relation to weak central coherence and event schemas ( 65 ) , and executive function ( 66 ) . taken together , these studies do point to consistent deficits in various aspects of tom in individuals with asd . social empathy is another area of research that has been widely investigated and related to atypical social interactions among individuals with asd . arguably , one of the first steps in social competency is the ability to be empathic . empathy can be categorized into two principle areas : cognitive empathy , which is the process by which one understands another s perspective , and is related to tom ( 67 ) , and emotional empathy , which is defined as an emotional response to another s emotional display ( 67 , 68 ) . while some children with high - functioning asd may have more advanced tom capabilities ( i.e. , cognitive empathy ) , the impairments in identifying emotions in others are still thought to be present ( 69 ) . specific deficits in empathy - related processes include the understanding of emotional stimuli , intersubjectivity , and reflecting of affect ( 70 - 73 ) , all of which , when deficient have the capacity to negatively impact social reciprocity . hence , the deficit in emotional empathy in individuals with asd may greatly impede social functioning . for individuals with asd , difficulties in emotion processing the implications of such deficits are many , not the least of which are emotional and physical isolation from family , peers and community . impairments in social functioning and the inability to understand emotion also commonly result in peer rejection and poor social support ; often producing higher levels of loneliness and poor quality of friendships for children with asd , in comparison to typically developing peers ( 13 , 74 ) . on self - report measures of friendship quality , children with asd endorse less security and trust in friendships , lower levels of companionship , and less helpfulness from friends ( 13 ) . while loneliness and understanding of friendship are closely linked for typically developing children , bauminger and kasari ( 2000 ) hypothesize that high - functioning children with asd may lack the affective glue to understand how to utilize their understanding of friendship to reduce loneliness . thus , the lack of affective bonding between children with asd and their peers limits the ability to initiate and maintain relationships . similarly , many adolescents and adults with asd also lack close friendships compared to typically developing peers ( 75 ) . possibly due to a rise in the complexity of social cues and a greater need for understanding of emotion processing that accompanies developmental maturity , social deficits often become even more prominent as children with asd enter adolescence ( 76 ) and adulthood ( 77 ) . although certain symptom aspects of asd may improve with time and intervention , social difficulties often related to the comprehension of emotions , seem to persist throughout the individual s lifespan and may represent a more chronic deficit in autism ( 11 , 78 ) . as adults , the lack of community connections and friendships that are often taken for granted by typically developing persons ( 75 ) may contribute to higher rates of depression , anxiety ( 79 ) , and victimization among individuals with asd ( 80 ) . thus , identifying and developing interventions to improve socio - emotional functioning that are of most help to individuals with asd is extremely important . in typical development , the sensation that we possess a kind of immediate understanding of what others are doing or feeling is so pervasive and automatic , that we rarely think about it . in fact , this connectedness often allows us to feel others emotions , as if they were our own . recently , neuroscience has provided a possible neural mechanism for this matching of sensory information to our own internal representations in the discovery of the mirror neuron system . researchers working with monkeys , discovered individual neurons in the macaque brain that fire both when an action is executed and when that same action is observed or heard ( 81 ) , leading these neurons to be called fadiga and colleagues ( 1995 ) provided the first demonstration of a neural system for coding the correspondence between observed and executed actions in humans using single - pulse transcranial magnetic stimulation ( tms ) . subsequent work then confirmed these findings , leading researchers to conclude that also in humans , there is a motor resonance system resembling the one described in the monkey ( 82 - 86 ) . these data showing that we use the same neural resources to represent the actions and emotions of others , as our own provide evidence for a neural substrate that can parsimoniously explain our intuitive feeling of effortlessly understanding others actions and emotions . using functional magnetic resonance imaging ( fmri ) to localize brain regions showing mirror properties , iacoboni and colleagues ( 1999 ) hypothesized that imitation , which contains both an observation and execution component would lead to a bold ( blood - oxygen - level dependent ; and indirect measure of neural activity ) signal increase greater than either action observation or execution alone . this study found two cortical areas that showed this predicted pattern of activity , the posterior inferior frontal gyrus ( pifg ) and the rostral sector of the inferior parietal lobule ( ipl ) ( 87 ) . thus , mirror resonance mechanisms subserved by the fronto - parietal mns are involved in action perception and performance , meaning that this neural system is recruited during both the execution of our own actions , and the observation of the actions of others . our ability to understand others actions and emotions , may thus be based in our ability to resonate with others on a motor level , a function that may in fact ground our ability to know about other minds ( 88 ) . although still in its early stages of study , the mns in humans has already been associated with a wide variety of higher - level functions in addition to action representation , including imitation and imitation learning ( 87 , 89 - 91 ) , intention understanding ( 92 , 93 ) , empathy and tom ( 94 - 96 ) , self - representation ( 97 - 99 ) , and the evolution of language ( 100 , 101 ) . these , and subsequent studies of the human mns , have greatly contributed to our understanding of the neural bases of perception - action mechanisms , social communication and emotion ( 81 , 102 , 103 ) . interestingly , these cognitive functions subserved at least in part by the mns - including imitation ( 96 , 104 ) , empathy and tom ( 104 , 105 ) , self - representation ( 105 ) and language ( 106 ) - have all been implicated as deficient in autism . in fact , dysfunction of the mns has been proposed as a possible cause of autism ( 96 , 107 - 109 ) . due to its importance in imitation , a dysfunction in the mns may lead to a failure to develop reciprocal social abilities including shared / joint attention , empathy and tom , as well as gestural recognition and language ( 96 ) . deficits in imitation during early childhood development may be at the root of many of the social deficits that manifest themselves during the later development of patients with autism ( 110 ) . the fundamental similarity between imitation and attribution of mental states is that both processes involve translating from the perspective of another individual to oneself . in belief attribution , one creates a representation of the other person s representation of the world in one s own brain , while the same process occurs during imitation with an action plan originating from another person being translated to one s own frame of reference ( 111 ) . autism - specific deficits in imitation have consistently been found ( 44 , 112 ) . the errors in imitation of patients with asd , seem to suggest that they have a deficit in the basic ability of translating action plans from the perspective of others to themselves ( 111 ) . differences in emotion processing between typically developing individuals and their peers with asd are also observable at a neural level . a recent neuroimaging study investigating imitation of emotional facial expressions has shown that children with autism have virtually no activity within the mns compared to typically developing controls , clearly linking their social isolation to a neural system important for understanding the intentions , actions , and emotions of others ( 113 ) . thus , the mns offers a potential neural substrate for understanding the social difficulties encountered by individuals with asd , while somewhat paradoxically , the mns may also offer a neural correlate for the special appreciation of music shown by individuals with asd . in contrast to the wealth of studies on emotion processing from body gestures and facial expressions of others , emotion processing in music is a relatively new area of research . music , like facial expressions , can convey emotions , and the ability to enjoy music is a universal human trait music also has the powerful ability to stimulate emotions , trigger memories , and intensify our social experiences . research in the psychology of music has begun to emphasize the intensely social nature of musical activities ( 114 ) , which may even hold the key to music s potential as a therapeutic and educational tool ( 115 ) . in fact , the last decade has seen an explosion of scientific research into the neural basis of music , revealing that different aspects of musical processing recruit almost all regions of the brain including prefrontal cortex , premotor cortex , motor cortex , somatosensory cortex , temporal lobes , parietal cortex , occipital cortex , cerebellum , and limbic regions including the amygdala and thalamus unlike any other stimulus or cognitive process ( 116 - 119 ) . it has also been shown that components of the limbic system involved in processing emotional stimuli in general the amygdala , the nucleus accumbens ( na ) , the hypothalamus , the orbitofrontal cortex ( ofc ) , the ventromedial prefrontal cortex ( vmpfc ) , the anterior cingulate cortex ( acc ) and the anterior insula ( ai ) - are also involved during the experience of both pleasant and unpleasant music ( 120 - 125 ) . this shows a neural overlap between emotion processing of non - musical stimuli ( e.g. pictures , faces ) and musical stimuli . in the classic paper first describing autism , leo kanner ( 1943 ) presented eleven case studies of children with autism and repeatedly mentioned musical abilities and musical interest in six of the children ( 126 ) . since then , researchers have systematically studied the musical processing abilities of individuals with autism , and have shown that while language abilities may be deficient ; individuals with asd seem to process music in similar ways to typically developing individuals ( 127 , 128 ) . moreover , individuals with asd appear to show a spontaneous preference for musical over verbal stimuli ( 129 ) , and it has been reported that approximately 40% of individuals within this population express a special interest in music ( 130 ) . these reports of prevalent interest in music within the asd population suggest that musical appreciation may be unimpaired in asd , and may even represent a particular ability area ( 126 , 131 ) . for example , applebaum and colleagues ( 1979 ) compared three musically nave children with asd and three typically developing musically experienced children on their ability to sing back musical stimuli . they found that children with autism performed as well or better than the musically experienced controls ( 132 ) . furthermore , a number of studies have indicated that individuals with asd show superior pitch abilities . for example , heaton and colleagues ( 1998 ) found that children with asd performed better than control children on a pitch memory task , while performing equally well on a speech sound memory task . in fact , children with autism remembered more tone / picture pairs one week after initial exposure than controls did after 2.5 minutes ( 133 ) ! more recently , heaton ( 2003 ) found that children with high - functioning autism showed enhanced pitch memory and labeling ( 130 ) . in studies of pitch discrimination , individuals with highfunctioning autism were superior to the comparison group , showing enhanced perception of local pitches ( 134 ) and exhibiting an increased sensitivity to pitch on both discrimination and categorization tasks ( 135 ) . individuals with asd not only show exceptional abilities in the domain of musical processing abilities , but research suggests that affective qualities of music might also be understood by this population . heaton and colleagues ( 1999 ) demonstrated that children with asd do not differ from typically - developing controls in their ability to perceive happy and sad affect in musical excerpts ( 127 ) . in a recent follow - up study , it was shown that active music listening is characteristic of children with autism , and that this listening results in the acquisition of culturally embedded knowledge about musical meaning . based on their findings , the authors conclude that emotion - processing in music may be preserved , and may be closely related to the level of language development , rather than asd diagnosis ( 128 ) . this apparently spared ability to understand the affective qualities of music is in marked contrast with the general difficulties in emotional communication and interpretation experienced by individuals with asd in other domains , such as in social communication ( 136 , 137 ) . how does emotional music affect the brain of individuals with asd ? bridging recent neuroimaging findings on the neural bases of intention understanding , action perception and social communication ( 90 , 93 , 138 , 139 ) with findings on the neural bases of music perception , it has been proposed that interactions between the mns and the limbic system may allow the human brain to understand complex patterns of musical signals and provide a neural substrate for the subsequent emotional response ( 115 , 140 ) . the shared affective motion experience ( same ) model of music perception holds that music is perceived not only as an auditory signal , but also as intentional , hierarchically organized sequences of expressive motor acts behind the signal ; and that the mns allows for corepresentation and sharing of a musical experience between agent and listener . musical sounds are imbued with intentional expression , evoking the sense of an intentional agent , and leading to the sense of a shared experience in the listener . within a neural network involving the temporal cortex , the fronto - parietal mns , and the limbic system , auditory features of the musical signal are processed primarily in the superior temporal gyrus and are combined with structural features of the expressive motion information within the mns . the anterior insula forms a neural conduit between the mns and the limbic system ( 94 ) , allowing incoming information to be evaluated in relation to the perceiver s autonomic and emotional state , thus leading to a complex emotional response to the music . the recruitment of these neural systems in both the agent ( musician ) and the listener allows for a shared affective motion experience ( same ) ( 115 ) . as a result , the expressive dynamics of heard sound gestures can be interpreted in terms of the expressive dynamics of personal vocal and physical gestures ( 115 , 140 ) . it might appear puzzling that children with asd seem to have deficits in the functioning of their mns , yet are able to experience some of the affective qualities of music . however , it is known that children with asd are particularly drawn to abstract patterns , and the repetitive , predictable nature of musical sounds may fulfill such a role . it is also possible that through experience and familiarity with these patterns , or indeed through shared musical imitation and synchronization activities , the mns may become sufficiently engaged for children with asd to move from the appreciation of musical sound patterns to the appreciation of the emotional state of the agent making them ; an agent who appears to behave in predictable , familiar ways that are comforting and companionable , rather than confusing . while the social world with its lack of predictability is often confusing and even frightening for the individual with asd , the predictable patterns typically found in musical stimuli may provide a reassuring and foreseeable avenue for emotion processing . it may also be highly relevant that music is dependent on auditory processing , since this might allow for attention to the socially salient information , whereas such socially salient information presented visually has been reported as unrewarding ( 49 ) and in some cases aversive ( 141 ) for individuals with asd . during the early development of music as a therapeutic tool , alvin ( 1975 ) and nordoff and robbins ( 1977 ) ( 142 - 144 ) referred to musical attunement being achieved between the therapist and the child the therapist would meet the child in music , and through this subtle , rhythmic interaction , communication was facilitated . it is within and through this shared , affective , musical experience that music therapy may have its greatest beneficial effect on socio - emotional behaviors . accordingly , decades of music therapy research have indicated that autistic children do respond well to music therapy ( 142 , 145 ) , as music facilitates and supports their desire to communicate ( 146 , 147 ) . trevarthen ( 2000 ) has proposed that the use of music therapy with autistic children may help improve their socio - emotional communication abilities , by helping them to develop timing and motor skills . brownell ( 2002 ) has also found that the use of musically adapted storytelling is an effective and viable treatment option for modifying target behaviors of children with autism ( 148 ) . while typically developing children often learn basic social rules and emotion processing through observation and imitation of peer behavior and/or specific instruction from caregivers ( 149 , 150 ) , children with asd often require further instruction . learning to navigate the social world is particularly challenging for persons with asd , since the natural development and transmission of necessary emotion processing and understanding of social cues requires generally positive and sustained interaction with others . initiating and sustaining joint attention , which is often absent in children with asd , is a primary therapeutic goal of music therapy . the same model proposes that by creating the feeling of being together - by jointly attending to music through imitation , synchronization , and shared affective experience - these powerful components of musical experience may be key aspects of therapeutic and educational intervention activities for individuals with asd ( 115 ) . several reviews of the literature have reported consistent and significant improvements in communicative behavior and emotional responsiveness by means of music interventions in individuals with asd ( 151 - 153 ) . while the nature of the musical interventions in individual studies is heterogeneous , a recent meta - analysis of ten studies comparing music to no - music conditions in the treatment of children and adolescents with autism , found an overall effect size of d = 0.77 , indicating a significant benefit of music intervention . some of the areas of improvement included : increased appropriate social behaviors and decreased inappropriate , stereotypical , and selfstimulatory behaviors ; increased verbalizations , gestures , and comprehension ; and increased communicative acts and engagement with others , among other positive effects ( 152 ) . as the above evidence illustrates , children with asd may benefit in particular from shared musical experience , as it offers opportunities for supporting the areas of social engagement and nonverbal communication in which they have some of their most profound difficulties ( 153 ) . thus , teaching the skills necessary to comprehend emotion processing has a significant life - long impact for persons with asd , highlighting the critical need for evidence - based interventions to assist these individuals in improving their social and emotion recognition skills . further research in the area of emotional music perception and music - based therapies may provide some clues about effective intervention strategies for the treatment of socio - emotional deficits in asd . perhaps the most fundamental level of social reciprocity is the ability to be in a relationship , which is founded upon the critical component of emotional attunement . the importance of affect exchange is evident from the earliest days of life , when social reciprocity begins with affect sharing and coordination with the caregiver . trevarthen and aitken ( 1994 ) have proposed that there is an intrinsic motive formation that acts as the foundation for the infant s adaptation to her / his environment . this pattern of neural activity works to direct the infant s brain emotionally as they self - regulate and try to understand the feelings of others ( 17 ) . these early exchanges of affect between caregivers and infants lay the foundation for the development of more complex types of communication later in life ( 18 ) . emotional attunement in typically developing children is documented as early as nine minutes of age , where infants preferentially attend to a face - like stimulus ( 19 ) , and demonstrate a preference for their mother s voice at birth ( 20 ) . infants as early as two to three months of age begin to discriminate between several different affective expressions shown by their parents ( 20 ) . at five months , infants are developing the ability to read affect in the faces of strangers and at six months are able to emotionally attune to their caregivers ( 21 ) . arguably , the foundation for emotional attunement and complex social interactions is dependent upon the emergence and development of joint attention . joint attention is defined as the ability to coordinate interest in external objects or events with other people ( 22 ) , and is thought to be an attempt to share the experience of objects and events with others ( 23 ) . among typically developing infants , the ability for joint attention emerges between 9 and 15 months ( 24 ) . this is when the infant learns to use gaze to direct the attention of others to objects of interest ( 25 ) . an inherent interest in other people appears to provide the motivation for some of the behavior that leads to learning to share and track gaze ( 26 - 29 ) . imitative behavior also becomes more prevalent , precise and object - directed at this time . for example , infants will typically imitate the ways in which adults manipulate objects ( 30 ) . studies have shown that beginning in early infancy , typically developing infants begin to use social referencing to make sense of confusing or threatening situations or persons , by looking at the face of their caregiver for information ( 31 , 32 ) . such behaviors that emerge around nine months and continue to develop into the third year of life increase the infants understanding of self and others as intentional agents , and are based in the gradual development of theory of mind ( 33 ) . theory of mind ( tom ) is the ability to understand the desires , intentions , and knowledge of another ( 34 ) . typically developing children generally show evidence of a tom around age four as they begin to understand the perspectives of other persons ( 35 ) . based upon a study examining non - verbal looking time using the violation - of - expectation method , it has been argued that the emergence of a tom with an implicit understanding of beliefs may even begin to develop as early as 15 months ( 36 ) . in examining precursors to tom , sodian and thoermer ( 2008 ) show that children under the age of three have the ability to use their own and others epistemic states in communication when engaging in false - belief tasks ( 37 ) . as children continue to grow , their ability to read affective cues and socially reciprocate becomes more refined . the gradual acquisition of such knowledge allows one to be part of and function successfully in a given culture , by incorporating others perspectives into one s own mind through cultural learning ( 33 ) . compared to typically developing children , the developmental trajectory of affect attunement is very different among infants with asd . even in infancy , children with asd fail to show the typical interest in human faces , instead showing a preference for inanimate objects ( 38 - 41 ) . the automatic mirroring of affective gestures and expressions is also considerably weaker in infants with asd ( 42 - 44 ) . consequences related to these deficits are displayed during infancy , and extend throughout childhood development . children with asd tend to pay less attention to the negative emotions of others compared to typically developing children ( 45 - 47 ) . they also appear to have more negative affective exchanges ( 48 ) , and display less positive emotion to their social partner ( 49 , 50 ) when compared to typically developing peers . the lack of attention paid to emotional cues often causes a rift in social interactions as well as lowering the affective responsivity of children with asd . emotional reciprocity is fundamental to the development of social skills and successful social interactions . due to the combination of deficits exhibited by children with asd in this realm , deficits in joint attention among individuals with autism are well - documented ( 51 - 53 ) . mundy , sigman and kasari ( 1994 ) found that joint attention for children with asd was lower than developmentally delayed and typically developing children ( 24 ) . in a more recent study , naber and colleagues ( 2007 ) examined different types of joint attention , including basic , associated , and visual . at 24 months , children with asd showed significantly less overall joint attention correlated with characteristics of autism and developmental level , but at 42 months the associations disappeared . of the three types of joint attention , only joint visual attention was less at 42 months , suggesting that joint visual attention may be the distinct deficit of concern in the development of joint attention in children with asd ( 54 ) . in research on gaze behavior it has been found that , compared to typically developing children , young children with asd shift attention between an object and another object more often than between a person and object or between person and person ( 55 ) . furthermore , children with asd spend less time looking at people and more time looking at objects , compared to neurotypical children ( 55 ) . by contrast , the processing of cartoons with human figures does not show any difference between typically developing children and children with asd ( 56 ) . in a study by klin and colleagues ( 2002 ) , it was found that when looking at faces , adults with asd looked at the eyes only half the time of neurotypical adults , but twice as often at mouths and objects ( 57 ) . a recent neuroimaging study found that brain responses to non - social stimuli ( houses ) were modulated by attention , both in adults with asd and a matched control group , but only the control participants demonstrated attentional modulation of face - selective regions during viewing of social ( face ) stimuli ( 58 ) . thus , the participants with asd demonstrated a lack of attentional modulation for the social stimulus . taken together , these findings suggest that atypical gaze patterns in individuals with asd may be related to the reduced salience of social stimuli from the earliest days of life , a deficit that persists into adulthood . indeed , reduced visual attention to socially salient stimuli may underpin many of the social communication impairments seen in individuals with asd . while typically developing infants use social referencing to make sense of confusing or threatening situations ( 32 ) , children with asd are less likely to reference another person in these same instances ( 59 ) - they are unlikely to voluntarily share their experience with others , and avoid initiating triadic interactions , even when guided to do so ( 60 ) . however , these atypical social behaviors in children with asd have been suggested to be secondary to impairments in the ability to represent mental states of others , or tom ( 61 ) . normal intellectual and verbal abilities appear to lessen the impact of earlier impairment in tom , such that in structured situations , individuals with asd may actually demonstrate tom abilities ( 62 , 63 ) . tom has been studied in relation to several other aspects of asd , including how individuals with asd are less likely to utilize tom to spontaneously attribute meaning ( 64 ) , in relation to weak central coherence and event schemas ( 65 ) , and executive function ( 66 ) . taken together , these studies do point to consistent deficits in various aspects of tom in individuals with asd . social empathy is another area of research that has been widely investigated and related to atypical social interactions among individuals with asd . arguably , one of the first steps in social competency is the ability to be empathic . empathy can be categorized into two principle areas : cognitive empathy , which is the process by which one understands another s perspective , and is related to tom ( 67 ) , and emotional empathy , which is defined as an emotional response to another s emotional display ( 67 , 68 ) . while some children with high - functioning asd may have more advanced tom capabilities ( i.e. , cognitive empathy ) , the impairments in identifying emotions in others are still thought to be present ( 69 ) . specific deficits in empathy - related processes include the understanding of emotional stimuli , intersubjectivity , and reflecting of affect ( 70 - 73 ) , all of which , when deficient have the capacity to negatively impact social reciprocity . hence , the deficit in emotional empathy in individuals with asd may greatly impede social functioning . for individuals with asd , difficulties in emotion processing are compounded by the inability to accurately perceive and understand emotional cues . the implications of such deficits are many , not the least of which are emotional and physical isolation from family , peers and community . impairments in social functioning and the inability to understand emotion also commonly result in peer rejection and poor social support ; often producing higher levels of loneliness and poor quality of friendships for children with asd , in comparison to typically developing peers ( 13 , 74 ) . on self - report measures of friendship quality , children with asd endorse less security and trust in friendships , lower levels of companionship , and less helpfulness from friends ( 13 ) . while loneliness and understanding of friendship are closely linked for typically developing children , bauminger and kasari ( 2000 ) hypothesize that high - functioning children with asd may lack the affective glue to understand how to utilize their understanding of friendship to reduce loneliness . thus , the lack of affective bonding between children with asd and their peers limits the ability to initiate and maintain relationships . similarly , many adolescents and adults with asd also lack close friendships compared to typically developing peers ( 75 ) . possibly due to a rise in the complexity of social cues and a greater need for understanding of emotion processing that accompanies developmental maturity , social deficits often become even more prominent as children with asd enter adolescence ( 76 ) and adulthood ( 77 ) . although certain symptom aspects of asd may improve with time and intervention , social difficulties often related to the comprehension of emotions , seem to persist throughout the individual s lifespan and may represent a more chronic deficit in autism ( 11 , 78 ) . as adults , the lack of community connections and friendships that are often taken for granted by typically developing persons ( 75 ) may contribute to higher rates of depression , anxiety ( 79 ) , and victimization among individuals with asd ( 80 ) . thus , identifying and developing interventions to improve socio - emotional functioning that are of most help to individuals with asd is extremely important . in typical development , the sensation that we possess a kind of immediate understanding of what others are doing or feeling is so pervasive and automatic , that we rarely think about it . in fact , this connectedness often allows us to feel others emotions , as if they were our own . recently , neuroscience has provided a possible neural mechanism for this matching of sensory information to our own internal representations in the discovery of the mirror neuron system . researchers working with monkeys , discovered individual neurons in the macaque brain that fire both when an action is executed and when that same action is observed or heard ( 81 ) , leading these neurons to be called fadiga and colleagues ( 1995 ) provided the first demonstration of a neural system for coding the correspondence between observed and executed actions in humans using single - pulse transcranial magnetic stimulation ( tms ) . subsequent work then confirmed these findings , leading researchers to conclude that also in humans , there is a motor resonance system resembling the one described in the monkey ( 82 - 86 ) . these data showing that we use the same neural resources to represent the actions and emotions of others , as our own provide evidence for a neural substrate that can parsimoniously explain our intuitive feeling of effortlessly understanding others actions and emotions . using functional magnetic resonance imaging ( fmri ) to localize brain regions showing mirror properties , iacoboni and colleagues ( 1999 ) hypothesized that imitation , which contains both an observation and execution component would lead to a bold ( blood - oxygen - level dependent ; and indirect measure of neural activity ) signal increase greater than either action observation or execution alone . this study found two cortical areas that showed this predicted pattern of activity , the posterior inferior frontal gyrus ( pifg ) and the rostral sector of the inferior parietal lobule ( ipl ) ( 87 ) . thus , mirror resonance mechanisms subserved by the fronto - parietal mns are involved in action perception and performance , meaning that this neural system is recruited during both the execution of our own actions , and the observation of the actions of others . our ability to understand others actions and emotions , resonate with others on a motor level , a function that may in fact ground our ability to know about other minds ( 88 ) . although still in its early stages of study , the mns in humans has already been associated with a wide variety of higher - level functions in addition to action representation , including imitation and imitation learning ( 87 , 89 - 91 ) , intention understanding ( 92 , 93 ) , empathy and tom ( 94 - 96 ) , self - representation ( 97 - 99 ) , and the evolution of language ( 100 , 101 ) . these , and subsequent studies of the human mns , have greatly contributed to our understanding of the neural bases of perception - action mechanisms , social communication and emotion ( 81 , 102 , 103 ) . interestingly , these cognitive functions subserved at least in part by the mns - including imitation ( 96 , 104 ) , empathy and tom ( 104 , 105 ) , self - representation ( 105 ) and language ( 106 ) - have all been implicated as deficient in autism . in fact , dysfunction of the mns has been proposed as a possible cause of autism ( 96 , 107 - 109 ) . due to its importance in imitation , a dysfunction in the mns may lead to a failure to develop reciprocal social abilities including shared / joint attention , empathy and tom , as well as gestural recognition and language ( 96 ) . deficits in imitation during early childhood development may be at the root of many of the social deficits that manifest themselves during the later development of patients with autism ( 110 ) . the fundamental similarity between imitation and attribution of mental states is that both processes involve translating from the perspective of another individual to oneself . in belief attribution , one creates a representation of the other person s representation of the world in one s own brain , while the same process occurs during imitation with an action plan originating from another person being translated to one s own frame of reference ( 111 ) . autism - specific deficits in imitation have consistently been found ( 44 , 112 ) . the errors in imitation of patients with asd , seem to suggest that they have a deficit in the basic ability of translating action plans from the perspective of others to themselves ( 111 ) . differences in emotion processing between typically developing individuals and their peers with asd are also observable at a neural level . a recent neuroimaging study investigating imitation of emotional facial expressions has shown that children with autism have virtually no activity within the mns compared to typically developing controls , clearly linking their social isolation to a neural system important for understanding the intentions , actions , and emotions of others ( 113 ) . thus , the mns offers a potential neural substrate for understanding the social difficulties encountered by individuals with asd , while somewhat paradoxically , the mns may also offer a neural correlate for the special appreciation of music shown by individuals with asd . in contrast to the wealth of studies on emotion processing from body gestures and facial expressions of others , emotion processing in music is a relatively new area of research . music , like facial expressions , can convey emotions , and the ability to enjoy music is a universal human trait music also has the powerful ability to stimulate emotions , trigger memories , and intensify our social experiences . research in the psychology of music has begun to emphasize the intensely social nature of musical activities ( 114 ) , which may even hold the key to music s potential as a therapeutic and educational tool ( 115 ) . in fact , the last decade has seen an explosion of scientific research into the neural basis of music , revealing that different aspects of musical processing recruit almost all regions of the brain including prefrontal cortex , premotor cortex , motor cortex , somatosensory cortex , temporal lobes , parietal cortex , occipital cortex , cerebellum , and limbic regions including the amygdala and thalamus unlike any other stimulus or cognitive process ( 116 - 119 ) . it has also been shown that components of the limbic system involved in processing emotional stimuli in general the amygdala , the nucleus accumbens ( na ) , the hypothalamus , the orbitofrontal cortex ( ofc ) , the ventromedial prefrontal cortex ( vmpfc ) , the anterior cingulate cortex ( acc ) and the anterior insula ( ai ) - are also involved during the experience of both pleasant and unpleasant music ( 120 - 125 ) . this shows a neural overlap between emotion processing of non - musical stimuli ( e.g. pictures , faces ) and musical stimuli . in the classic paper first describing autism , leo kanner ( 1943 ) presented eleven case studies of children with autism and repeatedly mentioned musical abilities and musical interest in six of the children ( 126 ) . since then , researchers have systematically studied the musical processing abilities of individuals with autism , and have shown that while language abilities may be deficient ; individuals with asd seem to process music in similar ways to typically developing individuals ( 127 , 128 ) . moreover , individuals with asd appear to show a spontaneous preference for musical over verbal stimuli ( 129 ) , and it has been reported that approximately 40% of individuals within this population express a special interest in music ( 130 ) . these reports of prevalent interest in music within the asd population suggest that musical appreciation may be unimpaired in asd , and may even represent a particular ability area ( 126 , 131 ) . for example , applebaum and colleagues ( 1979 ) compared three musically nave children with asd and three typically developing musically experienced children on their ability to sing back musical stimuli . they found that children with autism performed as well or better than the musically experienced controls ( 132 ) . furthermore , a number of studies have indicated that individuals with asd show superior pitch abilities . for example , heaton and colleagues ( 1998 ) found that children with asd performed better than control children on a pitch memory task , while performing equally well on a speech sound memory task . in fact , children with autism remembered more tone / picture pairs one week after initial exposure than controls did after 2.5 minutes ( 133 ) ! more recently , heaton ( 2003 ) found that children with high - functioning autism showed enhanced pitch memory and labeling ( 130 ) . in studies of pitch discrimination , individuals with highfunctioning autism were superior to the comparison group , showing enhanced perception of local pitches ( 134 ) and exhibiting an increased sensitivity to pitch on both discrimination and categorization tasks ( 135 ) . individuals with asd not only show exceptional abilities in the domain of musical processing abilities , but research suggests that affective qualities of music might also be understood by this population . heaton and colleagues ( 1999 ) demonstrated that children with asd do not differ from typically - developing controls in their ability to perceive happy and sad affect in musical excerpts ( 127 ) . in a recent follow - up study , it was shown that active music listening is characteristic of children with autism , and that this listening results in the acquisition of culturally embedded knowledge about musical meaning . based on their findings , the authors conclude that emotion - processing in music may be preserved , and may be closely related to the level of language development , rather than asd diagnosis ( 128 ) . this apparently spared ability to understand the affective qualities of music is in marked contrast with the general difficulties in emotional communication and interpretation experienced by individuals with asd in other domains , such as in social communication ( 136 , 137 ) . how does the brain perceive music ? how does emotional music affect the brain of individuals with asd ? bridging recent neuroimaging findings on the neural bases of intention understanding , action perception and social communication ( 90 , 93 , 138 , 139 ) with findings on the neural bases of music perception , it has been proposed that interactions between the mns and the limbic system may allow the human brain to understand complex patterns of musical signals and provide a neural substrate for the subsequent emotional response ( 115 , 140 ) . the shared affective motion experience ( same ) model of music perception holds that music is perceived not only as an auditory signal , but also as intentional , hierarchically organized sequences of expressive motor acts behind the signal ; and that the mns allows for corepresentation and sharing of a musical experience between agent and listener . musical sounds are imbued with intentional expression , evoking the sense of an intentional agent , and leading to the sense of a shared experience in the listener . within a neural network involving the temporal cortex , the fronto - parietal mns , and the limbic system , auditory features of the musical signal are processed primarily in the superior temporal gyrus and are combined with structural features of the expressive motion information within the mns . the anterior insula forms a neural conduit between the mns and the limbic system ( 94 ) , allowing incoming information to be evaluated in relation to the perceiver s autonomic and emotional state , thus leading to a complex emotional response to the music . the recruitment of these neural systems in both the agent ( musician ) and the listener allows for a shared affective motion experience ( same ) ( 115 ) . as a result , the expressive dynamics of heard sound gestures can be interpreted in terms of the expressive dynamics of personal vocal and physical gestures ( 115 , 140 ) . it might appear puzzling that children with asd seem to have deficits in the functioning of their mns , yet are able to experience some of the affective qualities of music . however , it is known that children with asd are particularly drawn to abstract patterns , and the repetitive , predictable nature of musical sounds may fulfill such a role . it is also possible that through experience and familiarity with these patterns , or indeed through shared musical imitation and synchronization activities , the mns may become sufficiently engaged for children with asd to move from the appreciation of musical sound patterns to the appreciation of the emotional state of the agent making them ; an agent who appears to behave in predictable , familiar ways that are comforting and companionable , rather than confusing . while the social world with its lack of predictability is often confusing and even frightening for the individual with asd , the predictable patterns typically found in musical stimuli may provide a reassuring and foreseeable avenue for emotion processing . it may also be highly relevant that music is dependent on auditory processing , since this might allow for attention to the socially salient information , whereas such socially salient information presented visually has been reported as unrewarding ( 49 ) and in some cases aversive ( 141 ) for individuals with asd . during the early development of music as a therapeutic tool , alvin ( 1975 ) and nordoff and robbins ( 1977 ) ( 142 - 144 ) referred to musical attunement being achieved between the therapist and the child the therapist would meet the child in music , and through this subtle , rhythmic interaction , communication was facilitated . it is within and through this shared , affective , musical experience that music therapy may have its greatest beneficial effect on socio - emotional behaviors . accordingly , decades of music therapy research have indicated that autistic children do respond well to music therapy ( 142 , 145 ) , as music facilitates and supports their desire to communicate ( 146 , 147 ) . trevarthen ( 2000 ) has proposed that the use of music therapy with autistic children may help improve their socio - emotional communication abilities , by helping them to develop timing and motor skills . brownell ( 2002 ) has also found that the use of musically adapted storytelling is an effective and viable treatment option for modifying target behaviors of children with autism ( 148 ) . while typically developing children often learn basic social rules and emotion processing through observation and imitation of peer behavior and/or specific instruction from caregivers ( 149 , 150 ) , children with asd often require further instruction . learning to navigate the social world is particularly challenging for persons with asd , since the natural development and transmission of necessary emotion processing and understanding of social cues requires generally positive and sustained interaction with others . initiating and sustaining joint attention , which is often absent in children with asd , is a primary therapeutic goal of music therapy . the same model proposes that by creating the feeling of being together - by jointly attending to music through imitation , synchronization , and shared affective experience - these powerful components of musical experience may be key aspects of therapeutic and educational intervention activities for individuals with asd ( 115 ) . several reviews of the literature have reported consistent and significant improvements in communicative behavior and emotional responsiveness by means of music interventions in individuals with asd ( 151 - 153 ) . while the nature of the musical interventions in individual studies is heterogeneous , a recent meta - analysis of ten studies comparing music to no - music conditions in the treatment of children and adolescents with autism , found an overall effect size of d = 0.77 , indicating a significant benefit of music intervention . some of the areas of improvement included : increased appropriate social behaviors and decreased inappropriate , stereotypical , and selfstimulatory behaviors ; increased verbalizations , gestures , and comprehension ; and increased communicative acts and engagement with others , among other positive effects ( 152 ) . as the above evidence illustrates , children with asd may benefit in particular from shared musical experience , as it offers opportunities for supporting the areas of social engagement and nonverbal communication in which they have some of their most profound difficulties ( 153 ) . thus , teaching the skills necessary to comprehend emotion processing has a significant life - long impact for persons with asd , highlighting the critical need for evidence - based interventions to assist these individuals in improving their social and emotion recognition skills . further research in the area of emotional music perception and music - based therapies may provide some clues about effective intervention strategies for the treatment of socio - emotional deficits in asd . in conclusion , research suggests that individuals with asd have marked difficulties with emotional communication and social interactions evidenced early in development by lack of emotional engagement and atypical patterns of inter - subjective behavior . however , individuals with asd do not appear to have difficulties recognizing simple emotions in music , in fact , they show an affinity for music , and may have some superior abilities with specific aspects of music processing . these findings imply a possible avenue for the development of more effective music - based therapeutic interventions to improve socio - emotional functioning in asd . the human mns , by creating a link at the neural level between two interacting agents ( e.g. the therapist and the patient ) , offers a neural substrate for music s therapeutic potential . however , empirical studies both neural and behavioral - are still required to further clarify the possible beneficial role of music for individuals with asd . in particular , behavioral studies that investigate the role of music in ameliorating social deficits , such as using musical cues to teach the meaning of other socially salient information , to individuals with asd will be crucial . neuroimaging studies will also be essential to help us understand how brain systems , such as the mns , which reportedly underlie social deficits in asd , can be activated by music to support social understanding . such future research has the potential to provide a doorway to evidence - based therapeutic interventions of great benefit to this population .
understanding emotions is fundamental to our ability to navigate and thrive in a complex world of human social interaction . individuals with autism spectrum disorders ( asd ) are known to experience difficulties with the communication and understanding of emotion , such as the nonverbal expression of emotion and the interpretation of emotions of others from facial expressions and body language . these deficits often lead to loneliness and isolation from peers , and social withdrawal from the environment in general . in the case of music however , there is evidence to suggest that individuals with asd do not have difficulties recognizing simple emotions . in addition , individuals with asd have been found to show normal and even superior abilities with specific aspects of music processing , and often show strong preferences towards music . it is possible these varying abilities with different types of expressive communication may be related to a neural system referred to as the mirror neuron system ( mns ) , which has been proposed as deficient in individuals with autism . music s power to stimulate emotions and intensify our social experiences might activate the mns in individuals with asd , and thus provide a neural foundation for music as an effective therapeutic tool . in this review , we present literature on the ontogeny of emotion processing in typical development and in individuals with asd , with a focus on the case of music .
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Proceed to summarize the following text: road - salt runoff poses an increasing threat to aquatic ecosystems that are influenced by urban land use and transportation corridors . four broad issues suggest that road - salt runoff is a serious and increasing threat to the nation s receiving waters . first , there is a multitude of historical evidence documenting detrimental effects of road salt on water chemistry and aquatic life . studies have continued each decade since then , with more comprehensive evidence of water - quality impacts from road salt . a small sampling of some representative topics studied includes specific water - quality impacts such as increased chloride and sodium concentrations , seasonality , climatic and land - use influence , vertical density gradients , and influence on sediment pore water , mixing and alteration of turnover in lakes ( 25 ) , and aquatic toxicity impacts ( 2,6,7 ) . second , road salt usage in the united states has increased steadily beginning in the 1940s through the current decade ( 8,9 ) . average annual salt sales in the united states for deicing purposes by decade beginning in 1940 were 0.28 ( 1940s ) , 1.1 ( 1950s ) , 4.1 ( 1960s ) , 8.7 ( 1970s ) , 8.8 ( 1980s ) , 13.0 ( 1990s ) , and 16.0 ( 20002008 ) million metric tons per year . third , urban development is increasing each year ( 10 ) , which increases the amount of impervious area on which winter deicing operations are conducted . this collective information suggests that the increasing road - salt usage trends of the previous seven decades will likely continue under current management conditions . fourth , chloride , and to a large degree sodium , the two primary ions in road salt , remain in solution , making it difficult with present - day technology to design effective management practices for reduction of road - salt loadings to receiving waters after application . currently , reduction in usage appears to be the only effective road - salt - runoff management strategy . the objective of this study was to investigate the influence of road - salt runoff on surface water and aquatic organisms . water - quality sampling was conducted on a local and regional scale . in the milwaukee metropolitan area ( local scale ) , streams were sampled for chloride , specific conductance , and aquatic toxicity to assess direct impact on aquatic organisms . in southern and eastern wisconsin ( regional scale ) , streams were monitored continuously for specific conductance , a surrogate for chloride , to assess potential impact on aquatic organisms . on a national scale , analysis of historical data was conducted for 17 metropolitan areas . data were retrieved from the u.s . geological survey ( usgs ) national water information system ( nwis ) for chloride concentrations from streams sampled between 1969 and 2008 . environmental protection agency ( usepa ) water - quality criteria and analyzed for seasonal differences . twelve streams in the milwaukee metropolitan area and one reference stream north of milwaukee were sampled in february and march 2007 for determination of water chemistry and aquatic toxicity ( table 1 , figure 1 ) . twelve of the streams had substantial urban land - use contribution and the reference stream had 80% natural areas and no urban land use ( parnell creek ) . drainage areas of these streams ranged from 16.4 km ( 6.33 mi ) at willow creek to 1833 km ( 872 mi ) at the milwaukee river ( table 1 ) . a 14th stream , wilson park creek , was monitored selectively from 1997 through 2007 during deicing periods . sample results from these 14 streams include chloride concentrations , specific conductance values , and bioassays using pimephales promelas and ceriodaphnia dubia . location of study sites in wisconsin and metropolitan areas in the united states used for aquatic toxicity evaluation from road salt . eleven streams in southeast wisconsin were monitored using continuous specific conductance sensors with resulting data used as an indication of road - salt runoff ( table 1 , figure 1 ) . these streams represent a gradient of land use with urban influence ranging from 6.0 to 100% . individual water - quality samples for chloride in 17 major metropolitan areas around the country were retrieved from nwis , the u.s . candidate streams were selected based on the latitude and longitude of the monitoring location and its proximity to major urban land - use areas . streams ultimately chosen for this study included those that were sampled for chloride between 1969 and 2008 , had at least 12 samples in the cold - weather months ( november to april ) , 12 samples in the warm - weather months ( may to october ) , and a drainage area of less than 2600 km . a total of 12 005 samples from 162 sites in the northern part of the united states and 2378 samples from 50 sites in the southern part of the united states ( south of st . for the 13 milwaukee area streams , sampling periods were targeted at runoff events during road - salt application periods . real - time specific conductance data from wilson park creek were used as an indicator of road salt in milwaukee area streams and used to initiate this sample collection . water - quality samples were collected manually during the february 26 and march 7 , 2007 sampling periods . samples were collected either by submerging sample bottles directly into the center of the channel for wadeable streams or by using a weighted - bottle sampler to collect cross - section integrated samples from a bridge for nonwadeable streams ( 11 ) . comparison of the relation between chloride and specific conductance was used to assess potential bias in results . all samples were within 10% of the resulting linear regression except those with chloride concentrations less than 230 mg / l , where chloride and sodium were a less dominant influence on specific conductance . flow - weighted composite samples were collected at wilson park creek from 1997 through 2007 . specific details of the sampling protocol used from this site have been previously published ( 12 ) . weather data were retrieved from three nearby noaa weather stations ( general mitchell international airport , mount mary , and germantown ) . on february 24 , 25 , and 26 , 2007 , average snowfall was 16 , 15 , and 2 cm ( 0.9 , 1.7 , and 0.2 cm water equivalent ) , and maximum air temperatures were 0.5 , 2.8 , and 2.8 c , respectively . on march 7 , there was an average of 5.7 cm of snow ( 0.4 cm water equivalent ) , and maximum air temperature of 0.5 c . this was not enough snow to trigger general plowing ; however , salt was applied on paved surfaces to melt snow and ice . salt application and temperatures greater than 0 c for both of these events resulted in runoff from impervious areas flowing to storm sewers and receiving streams . measurements from continuously deployed specific conductance sensors were recorded at least every hour and as frequently as every 5 min depending on the individual site and specific hydrologic conditions . instantaneous specific conductance was measured in the 13 milwaukee area streams at the time of the 2007 sampling periods . all specific conductance sensors were maintained in accordance with standard usgs methods ( 13 ) . chloride and toxicity tests were conducted at the wisconsin state laboratory of hygiene using well established methods that are presented in the supporting information . toxicity was exhibited in samples from 7 of the 12 urban - influenced watersheds in the milwaukee metropolitan area that were collected during road - salt application periods in february and march , 2007 ( figure 2 ) . adverse response in c. dubia tests occurred in samples with chloride concentrations of 1610 mg / l or greater . adverse response in p. promelas tests occurred in samples with chloride concentrations of 2940 mg / l or greater . the ic25 values ( the concentration at which there is 25% inhibition ) computed using measured chloride concentrations in these stream samples were 1050 mg / chloride concentrations were elevated above the epa acute water - quality criteria concentration of 860 mg / l in eight of these samples and above the epa chronic water - quality criteria concentration of 230 mg / l ( 14 ) in 11 of these samples , indicating potential for aquatic toxicity effects . a sample collected at the rural reference site during the february sampling period had a chloride concentration of 20.4 chronic bioassay results in relation to chloride concentration in samples collected from 13 streams in the milwaukee , wi metropolitan area , februarymarch , 2007 : ( a ) c. dubia survival and mean young produced , and ( b ) p. promelas survival and mean weight . specific conductance results from continuous monitoring in wilson park creek in milwaukee during 2007 indicate that conditions similar to the february and march 2007 sampling periods were common during the cold - weather period of 2007 ( figure 3 ) . specific conductance in wilson park creek in milwaukee , wi during 2007 in reference to aquatic toxicity sampling periods ( triangles ) for 13 milwaukee area streams . results from 37 samples collected from 1997 to 2007 at wilson park creek in milwaukee demonstrate long - term toxicity effects in numerous samples and a distinct relation to chloride concentration ( figure 4 ) . concentrations at which chronic result effects were observed from this long - term sampling program were very similar to corresponding concentrations where chronic effects were observed from the 2007 sampling events in the milwaukee metropolitan area . in chronic c. dubia assays , mg / l or greater ( 43% of samples ) and complete mortality was observed at chloride concentrations of 2420 mg / l and greater ( 38% of samples ) . initial toxic effects began between 600 and 1100 mg / l . mortality was observed in acute c. dubia assays for all samples with chloride concentrations greater than 1900 mg / l . in chronic p. promelas assays , reduced weight and survival was present when concentrations were 2920 mg / l or greater . results for both of these organisms in chronic bioassays indicate scatter or uncertainty with lower chloride concentrations . this could be due to variability in the actual test , confounding contaminants in urban runoff , or a combination of these factors . it was difficult to determine the exact concentration at which road - salt effects began for chronic c. dubia assays due to this variability . in p. promelas acute assays , only two samples were influenced with initial effects occurring between 4660 and 6290 bioassay results in relation to chloride concentration in samples collected from wilson park creek in milwaukee , wisconsin , 19972007 : ( a ) c. dubia survival and mean young produced in chronic bioassays , ( b ) p. promelas survival and mean weight in chronic bioassays , ( c ) c. dubia survival in acute bioassays , and ( d ) p. promelas survival in acute bioassays . eleven streams in urban regions of wisconsin were monitored with continuous specific conductance sensors during cold- and warm - weather periods from 1998 to 2008 ( table 1 ) . between 1 and 10 years of data were available depending on the individual site . linear regression was conducted from results of concurrent analysis of chloride and specific conductance in samples from these streams . to reduce negative bias in residuals when other ions influence this relation at low concentrations ( specific conductance < 1400 s / cm ) , data in the regression this resulted in a slope of 0.374 and intercept of 328 ( r = 0.997 , supporting information figure s1 ) . this regression is used for the remainder of this paper to provide chloride concentration estimates ( referred to as clest ) from measurement of specific conductance . the maximum observed specific conductance in these streams increased with increasing urban land use ( figure 5 ) . the maximum clest for seven of these sites exceeded the usepa acute water - quality criteria value of 860 mg / l . mg / l at each of the 11 sites with a maximum clest of 289 mg / l for the least impacted stream . maximum specific conductance compared to urban land - use percentage in 11 wisconsin streams with reference to u.s . environmental protection agency water quality criteria for chloride ( 19 ) . the highest continuous specific conductance results at these eleven sites occurred during cold - weather months ( figure 6 ) . the most dramatic impacts from road - salt runoff were observed in the two highly urban watersheds lincoln and wilson park creeks in milwaukee with specific conductance often exceeding 10 000 s / cm ( clest = 3410 mg / l ) and at times exceeding 20 000 s / cm ( clest = 7150 mg / l , figure 6a ) . maximum monthly specific conductance at four sites with intermediate influence ranged between 3000 and 8000 s / cm ( figure 6b ) . maximum monthly values at four sites with low influence were still substantially impacted by chloride in cold - weather months , but maximum monthly specific conductance was less than 3000 s / cm ( figure 6b ) . although most of these watersheds were small to medium in size with a drainage area of 25280 km , the milwaukee river at milwaukee has a drainage area of 1800 km and still was impacted by road - salt runoff with a maximum specific conductance of 2850 s / cm . in all months , the average monthly maximum specific conductance was greatest in the sites with urban land use of 98% or greater , followed by those with 2669% urban land use , and least in sites with less than 26% urban land use ( table 1 , figure 6 ) . monthly maximum specific conductance from continuous monitoring at 11 sites in wisconsin over a gradient of urban influence . in some cases , specific conductance decreased through the warm - weather months , reaching a minimum in october ( figure 6c ) . specific conductance in the two highly urban watersheds decreased from may through october by 34% and 39% . the average monthly maximum in these two streams was greater than 1200 s / cm throughout the entire year . specific conductance data from oak creek ( 63% urban land use ) also decreased by 26% from may through october . other sites either did not have sufficient data to evaluate warm - weather conditions or did not exhibit this effect . usgs chloride sample results from streams near metropolitan areas were retrieved from 1969 to 2008 for assessment of potential road - salt influence throughout the country and to provide context for the more intensive wisconsin study results ( figure 7 ) . the maximum number of sites per metropolitan area was 29 ( denver ) and the maximum number of samples per metropolitan area was 1690 ( cleveland ) . comparison of chloride concentrations to chronic ( a ) and acute ( b ) usepa water - quality criteria for warm - weather months and cold - weather months in streams from northern and southern urban areas . bars indicate the percent of sites for each metropolitan area that had at least one sample result greater than the water - quality criteria . a total of 898 samples were collected and analyzed for chloride at 21 monitoring locations within the milwaukee area . results exceeded 230 mg / l chloride in at least one sample at 90% of monitoring sites during cold - weather months and 33% of monitoring sites during warm - weather months ( figure 7a ) . similarly , 57% of these monitoring sites exceeded 860 mg / l chloride in at least one sample during cold - weather months , and none during warm - weather months ( figure 7b ) . most northern metropolitan areas included in the analysis demonstrated the same pattern as the milwaukee area sites . a total of 51% of all 168 northern monitoring locations had at least one sample with concentrations exceeding 230 mg / l during cold - weather months and 15% exceeded that concentration during warm - weather months . a total of 23% of northern monitoring locations had at least one sample with concentrations exceeding 860 mg / l during cold - weather months and 1% exceeded that concentration during warm - weather months . ten of 13 metropolitan areas had more monitoring sites that had a chloride sample result exceeding 230 mg / l during cold - weather months than during warm - weather months . nine metropolitan areas had more monitoring sites with sample results that exceeded 860 mg / l during cold - weather months than during warm - weather months . l during warm - weather months . at monitoring locations in the four southern metropolitan areas , few samples exceeded the water - quality criteria concentrations and no common seasonal pattern was detected . only 2 and 4% of monitoring locations had samples exceeding 230 mg / l during warm- and cold - weather months , respectively ; samples from the southern sites did not exceed 860 mg / l . several other southern metropolitan areas were analyzed but not included in this study because monitoring locations either had insufficient data or marine influence . detrimental impacts from road - salt runoff to surface water presented in this study were evident on local , regional , and national scales . the presented long- and short - term runoff sampling programs in wisconsin demonstrate a substantial effect from road salt on streamwater quality and aquatic life . bioassay results from runoff events confirm that the observed high concentrations of road salt caused acute and chronic toxicity to aquatic organisms . in addition , continuous specific conductance results indicate that elevated levels of road salt were present multiple times per year each year of monitoring . populations of aquatic organisms in these streams and others with such road - salt influence are likely limited to salt - tolerant species . effects on aquatic organisms have previously been demonstrated using a salt tolerance biotic index ( chloride contamination index , cci ) in toronto area streams ( 15 ) . when compared to a review published by environment canada including laboratory and field assessments of road salt impact on aquatic species , concentrations observed in the present research indicate that detrimental effects would be present over numerous taxa in addition to the organisms used in this study ( 7 ) . this review of road salt effects concluded that high concentrations may have immediate or long - term ecosystem population effects , and that lower levels of increased chloride concentrations may affect community structure , diversity , and productivity ( 7 ) . results from continuous specific conductance monitoring in lincoln and wilson park creeks in milwaukee indicate that elevated levels of chloride in these streams were common for extended periods of time , even through the summer months . similar to results from lincoln and wilson park creeks in milwaukee , study of groundwater influence on stream chemistry in massachusetts confirmed that chloride from highway - deicing applications persisted throughout the year as a source of contamination in groundwater , interflow , and streamwater even during warm - weather months ( 16 ) . the analysis of historical chloride data from urban areas around the country indicated potential for considerable and widespread impact from road salt on surface water quality and aquatic life . despite the limitation that sample results from these selected areas were from numerous studies not necessarily designed to capture periods of road - salt runoff , the influence of road salt was clear . streams with urban influence throughout the country in areas where road salt is applied are at risk for substantial contamination and detrimental effect on aquatic life . impacts of road salt are present in other geographic regions that commonly apply these deicers as well . for instance , in a study of urban streams in finland , chloride concentrations during road - salt runoff periods varied over 9-fold within one day with nacl concentrations from weekly composite samples observed as high as 567 mg / l ( 17 ) . salinity in these streams increased in direct proportion to accumulated application of road salt ( 18 ) . research on the influence of urban land use on aquatic life in streams has identified a level of 712% impervious surface where decreases in biological integrity were observed ( 1921 ) . much work investigating aquatic life degradation has focused on ambient water chemistry , habitat , and other physical , hydrologic , and hydraulic factors ( 22 ) . the relation of chloride concentrations and specific conductance with urban land use shown in this study and a recent study of the northern united states ( 23 ) indicates that road - salt runoff and other anthropogenic uses of chloride are important factors in the biological integrity of urban streams in the northern united states . although chloride sampling has been included in previous evaluations of urban streamwater quality ( 21 ) , water - quality sampling did not specifically focus on periods of winter runoff and may not fully represent the severity of road - salt influence . to better understand the relation between urban land use and stream biology , focused monitoring would be needed to characterize the range of chloride concentrations and duration of road - salt influence in streams during deicing periods . however , because of the episodic nature of road - salt runoff , the full range of in - stream road - salt influence is difficult to characterize without use of continuous- and event - based monitoring focusing on deicing periods . a periodic or fixed - interval sampling plan will not fully characterize road - salt influence except by happenstance . environmental management or mitigation of this issue solutions would require consideration of environmental , political , economic , and safety issues . added to this complexity is the diversity of applicators in urban areas . city maintenance crews de - ice roadways , public parking lots , and sidewalks while a host of private applicators de - ice commercial , institutional , and industrial areas , and homeowners apply de - icers to residential driveways and sidewalks . for example , use of organic salts such as calcium magnesium acetate would reduce chloride loading , but may increase biochemical oxygen demand thereby increasing potential for oxygen depletion in receiving waters . greater aquatic toxicity and water - quality impacts seem likely if increasing trends in road - salt usage and expanding urban development continue . regardless of methods chosen , reducing the impact of road salt on the environment would take a substantial and sustained effort coupled with consideration of numerous interconnected factors .
a new perspective on the severity of aquatic toxicity impact of road salt was gained by a focused research effort directed at winter runoff periods . dramatic impacts were observed on local , regional , and national scales . locally , samples from 7 of 13 milwaukee , wisconsin area streams exhibited toxicity in ceriodaphnia dubia and pimephales promelas bioassays during road - salt runoff . another milwaukee stream was sampled from 1996 to 2008 with 72% of 37 samples exhibiting toxicity in chronic bioassays and 43% in acute bioassays . the maximum chloride concentration was 7730 mg / l . regionally , in southeast wisconsin , continuous specific conductance was monitored as a chloride surrogate in 11 watersheds with urban land use from 6.0 to 100% . elevated specific conductance was observed between november and april at all sites , with continuing effects between may and october at sites with the highest specific conductance . specific conductance was measured as high as 30 800 s / cm ( cl = 11 200 mg / l ) . chloride concentrations exceeded u.s . environmental protection agency ( usepa ) acute ( 860 mg / l ) and chronic ( 230 mg / l ) water - quality criteria at 55 and 100% of monitored sites , respectively . nationally , u.s . geological survey historical data were examined for 13 northern and 4 southern metropolitan areas . chloride concentrations exceeded usepa water - quality criteria at 55% ( chronic ) and 25% ( acute ) of the 168 monitoring locations in northern metropolitan areas from november to april . only 16% ( chronic ) and 1% ( acute ) of sites exceeded criteria from may to october . at southern sites , very few samples exceeded chronic water - quality criteria , and no samples exceeded acute criteria .
You are an expert at summarizing long articles. Proceed to summarize the following text: since 2007 , ecuador has created a system of free , universal health care with government - funded primary care centers in low - income neighborhoods throughout the country . there is overwhelming demand for primary care services at these community health centers , and patients often experience long waits for their clinic visits . moreover , because clinic visits are necessarily brief , clinicians often do not have sufficient time to address all patient concerns and needs . such time constraints pose a significant challenge to providing patient - centered care . in such contexts , it is critically important for patients to be well prepared for their primary care clinic visits , with a clear idea of the principal questions and concerns they would like to address . research conducted in the united states using videotaped primary care encounters has shown that the first item addressed during the primary care visit receives the bulk of the attention ( 5 minutes versus 1.1 minutes for the next five out of six total topics addressed during the typical 15-minute visit ) . other visit content analyses have found that the likelihood of preventive screening declines with each additional concern brought up by patients , and more complex patients are less likely to have medication dose changes . the number and complexity of health issues and comorbidities a patient has as well as any behavioral , emotional , and social issues they face all added to the challenges of adequately addressing all of a patient 's key concerns and needs [ 5 , 6 ] . low - income ecuadorian adults with chronic illnesses often have low health literacy and face multiple social and other challenges to managing their conditions . accordingly , effective ways to better enhance their ability to prepare to best convey to their provider their principal concerns for a primary care visit are especially needed . public primary health care centers in ecuador , such as those serving the low - income communities of southern quito , currently do not have any kind of previsit form for patients . previsit forms can help patients identify their principal health concerns and questions , list their current prescribed medications and known medical conditions , and provide other basic information to help their providers prioritize patient concerns and topics to be addressed during office visits . in spite of long preclinic visit waits , patients currently do not have any preclinic visit preparation and do not complete previsit forms . due to the high numbers of patients who often have long clinic waits for appointments at the local health centers , clinic waiting rooms are an ideal setting in which brief interventions are delivered to patients while they wait for care . the basic concept of encouraging patients to prepare for their physician appointments is not new [ 812 ] . a number of studies have explored the impact of interventions and brief coaching on patient involvement and outcomes during clinic visits [ 1215 ] . one pilot study found that sharing personalized , clinical information with patients before clinic visits improved doctor / patient communication and increased patient participation without increasing the length of visits . however , a systematic review by kinnersley and colleagues ( 2008 ) found that previsit interventions to promote question asking only provide a small benefit to patients , possibly because it is difficult to change established norms between patients and physicians during these encounters . more research is needed on how to optimally help prepare patients , especially low - income patients with low health literacy who face multiple barriers to chronic disease management , for health care visits [ 9 , 10 , 16 ] . truly patient - centered care requires that care interactions include consideration of patient priorities and preferences [ 1719 ] . to address this need , we evaluated the effectiveness of a written previsit form for adults with chronic conditions to complete while waiting for their clinic visit to help prepare them to ask questions and provide information on their medical conditions and concerns to physicians . because patients with little formal schooling may face literacy obstacles to completing a written form on their own , we also sought to assess outcomes from providing assistance to patients to complete the form . we hypothesized that patients who completed a brief written form on their own and patients who completed the form with the help of research staff before the clinic appointment would be more likely to report having their main health concerns and questions addressed in the subsequent clinic visit and thus improved satisfaction with the visit compared to patients receiving usual care . if we found improved outcomes , we could then make the form available to the clinic for continued use after the intervention . we conducted a parallel , randomized controlled trial ( rct ) with scheduled patients waiting to see their primary care physicians in two community health centers serving indigent to low - income communities in southern quito from june 2011 through august 2011 . the patients were randomized to one of three arms : ( 1 ) usual care with no preclinic visit preparation ; ( 2 ) provision of a brief written form for patients to complete on their own ; or ( 3 ) provision of the written form with assistance from a member of the research staff in completing the form . the study received irb approval from the university of michigan , the pontifical catholic university in quito , ecuador , and the ministry of health in ecuador . when adult patients arrived at the clinic for their appointment , they were told that the clinic was evaluating patients ' experiences with clinic service and possible ways to improve service . they were then asked if they had any chronic conditions for which they had taken medications regularly for 3 months or longer to screen out patients who were recently diagnosed or were not getting regular care at the clinic . if they met this criterion , they were asked if they were willing to talk to a study team member to consider participating in an intervention study . if they agreed to participate , a study team member proceeded with oral informed consent and administered orally a brief baseline survey . in order to avoid contamination , the randomization was assigned weekly with each center carrying out one of the three study group conditions daily . depending on which of the three arms the clinic session had been randomized to that day , participants received no previsit form ( usual care ) ( group 1 ) , a written previsit form to complete alone with brief oral instructions from a team member ( group 2 ) , or a previsit form to be completed with the help of a team member ( group 3 ) . in group 3 the staff member reviewed the questions on the form orally with the patient and was available to help with form completion and questions while the participant was completing the form . this active assistance in completing the form took on average 510 minutes , and the most common questions concerned explanations of the questions on medication side effects , diet , and exercise . the research staff practiced their approach to providing oral assistance together before beginning the intervention to ensure uniformity across different staff . after their clinic visit , patients in all three groups were given the same instructions to complete a postclinic visit questionnaire administered orally by one of the study team . the study did not involve invasive procedures , medical recommendations , or review of medical records or other health record data . the previsit form encouraged patients to think about what they wanted to achieve during the visit . it included a list of medical conditions for patients to check off for past medical history . it also included a list of signs and symptoms that patients could check off if they were experiencing any at the time of their visit . technical terminology was avoided , and local colloquial terms were used ( e.g. , coto , vinagreras ) to facilitate patients ' understanding . there was a section for the patients to write in medical allergies and the list of their current medications , including prescription , over - the - counter , or any natural remedies . below the list of medications , we asked if they were experiencing any side effects from the medication and if for any reason they had stopped taking one of their medications in the last 6 months . the form asked patients to write their chief concern they wanted to address in the visit and provided a space for them to list the questions and concerns they had for that day 's clinic visit . to encourage patients to write down questions , examples were given such as drug side effects . participants were also encouraged to show the form to their providers during the clinic visit ( see appendix a in the supplementary material available online at http://dx.doi.org/10.1155/2015/724245 for previsit form ) . the baseline survey included age , gender , self - reported health status , education level , health literacy , comorbid conditions , chronic disease self - efficacy , level of patient activation , and understanding of and adherence to medications . the survey also included patients ' self - reported evaluations of their diet and exercise . we examined three main study outcome measures from a survey orally administered immediately after participants ' clinic visit . are you completely satisfied with your clinic visit today ? ; ( 2 ) whether the participants achieved all that they had wanted to during the doctor 's visit today ; and ( 3 ) whether the participants felt confident about asking questions to the doctor in the clinic visit ( see appendix b in the supplementary material for the baseline survey in spanish and appendix c in the supplementary material for the postvisit survey in spanish ) . we also examined differences between group 2 ( previsit form only ) and group 3 ( previsit form and assistance ) in whether participants wrote down at least one question or concern that they wanted to discuss with their doctor during the upcoming clinic visit and mean number of questions or concerns listed on the form . to determine differences in self - reported measures among the three groups , our recruitment goal was approximately 200 participants ( approximately 65 participants per group ) . the xtmixed command in stata , version 12.0 ( statacorp , college station , tx ) , which fits multilevel mixed - effects linear regression models , was used to examine intervention effects . consenting participants were randomly assigned to one of three groups : 64 were randomly assigned to the first group ; 68 to the second group ; and 67 to the third group . of the 199 patients enrolled , 187 ( 94% ) completed the post survey assessments . table 1 shows characteristics of participants who completed the initial survey ( n = 199 ) and assessment results . figure 2 shows changes in study measures for participants between baseline and the postclinic visit measures and compares postclinic visit assessments among group 1 , group 2 , and group 3 . in analyses adjusting for gender , patients who received the revisit form along with assistance from a team member ( group 3 ) were more than twice as likely as participants in group 1 to report that they had achieved everything they wanted during their visit ( aor 2.2 , p = 0.039 ) . while not statistically significant ( p = 0.071 ) participants in group 3 were 1.86 times as likely as group 1 to report that they felt capable in their clinic visit of asking questions . although a higher percentage of participants in group 3 reported greater levels of satisfaction with their clinic visit than participants in group 1 , this difference also was not statistically significant . there were no differences between groups 1 and 2 in any of our study outcome measures . figure 3 shows the comparison between the number of questions or concerns participants who received assistance in completing the form ( group 3 ) wrote down to discuss with their physician at the upcoming clinic visit compared to the number among participants who were just given the form to complete alone ( group 2 ) . patients who received help from a team member were more likely to write down at least one question ( 80% of patients in group 3 wrote down at least one question or concern compared with 50% in group 2 , p < 0.01 ) . patients in group 3 wrote down a mean of 1.6 questions compared to those in group 2 who wrote down a mean of 1.1 questions . among this sample of low - income ecuadorian adult patients with very low levels of formal education and at least one chronic condition requiring medication at a government - funded community health center in southern quito , patients who received assistance in completing a previsit form were more likely to engage in the consultation and to report accomplishing all their primary care visit goals and feeling capable of asking their physician questions at that visit than participants who did not receive any form or assistance . they were not , however , more likely to report feeling completely satisfied with their clinic visit than participants in the other groups . there were no differences in any measures between the group who received a previsit form but no assistance in completing the form and the group receiving no previsit forms or assistance . our findings reinforce evidence from observational and nonrandomized studies that suggest health benefits from receiving assistance in formulating questions and concerns for a clinic visit before the visit [ 912 , 16 ] . our findings also add to the literature by emphasizing ways such assistance may need to be adapted to meet the needs of low - literacy populations with low levels of formal education such as those served by many community health centers in low - income communities . assistance in completing a previsit form appeared to be more effective than receipt of a written form alone in improving some postclinic visit outcomes , but not all outcomes . because group 3 combined assistance completing a previsit form with receipt of the form , we can not determine whether orally eliciting patients ' questions and concerns might have led to similar benefits without completion of the previsit form . further research should explore reasons why providing the previsit form alone was not more effective than no previsit form and whether a different , simpler form could be effective . one possibility is that providing previsit forms alone is not effective among populations with high rates of low literacy . in our study sample , approximately 23% of participants in each group reported that they did not feel comfortable with filling out forms alone , and over 40% had only completed some primary school . of note , however , these variables were not associated with whether patients wrote down at least one question or concern in group 2 , the group that received the previsit form alone . it is also worth noting that while most primary care practices in the united states routinely have patients complete revisit forms of some sort , there is little evidence from the united states or europe on how completing previsit forms alone influences the subsequent primary care visit and discussion . there are , however , studies showing that providing cancer patients with question prompt sheets before oncology visits increases the number of questions patients ask in those visits [ 2628 ] . community health centers in southern quito thus , even though it only took 510 minutes to provide this assistance , different modalities to provide previsit coaching or assistance in formulating questions and concerns should also be explored . for example , as all the health centers in southern quito have television screens and the capacity to show dvds on these screens , it might be possible to develop an instructional dvd that could be shown in the waiting rooms to provide guidance to patients to think of questions and concerns they would like to discuss with their physician and to help in understanding how to fill out a previsit form . first , we only conducted a single preclinic brief intervention and only assessed participant - reported outcomes immediately after the clinic visit . future studies should test previsit assistance interventions in this population over longer periods and include clinical outcomes along with patient - reported outcomes . second , although we randomized clinic sessions to prevent contamination among the patients waiting to see the providers , the nature of the intervention prevented blinding of nurses and providers to the intervention itself . moreover , it is possible that having patients in some clinic sessions bringing in the previsit form with listed questions and concerns may have influenced physician behaviors with patients who did not complete previsit forms . if anything , however , this would have introduced bias that would have reduced any differences between study groups . third , this intervention focused exclusively on activating patients to discuss questions and concerns with their providers and did not address providers . research by chew - graham and colleagues ( 2013 ) analyzing primary care visits for long - term health conditions found that patient concerns were often missed or disregarded by providers during clinic visits . a more powerful intervention would also target provider behaviors to acknowledge and address patient concerns and questions they listed to be addressed at the clinic visit . the providers that participated in this intervention had a very favorable response to the previsit forms but were not given any guidelines on how they should use the forms during the visit , and no data was collected on how the forms influenced their behavior . another significant limitation is that the form started with targeted questions about medical conditions , medications , and adherence before asking about more general questions and concerns the patient had for that clinic visit . this ordering and predominance of medical questions may have unintentionally restricted patients in the concerns they felt comfortable to bring up and failed to adequately encourage them to raise their own concerns that may have extended beyond a medical focus . finally , we conducted this study among a predominantly female adult patient population in only two primary care clinics in one region of quito . in conclusion , our study suggests that providing assistance with completing a previsit form designed to help patients formulate questions and concerns to discuss with their provider improved their confidence in asking questions and belief that they had achieved all their visit goals . our study findings , however , do not support the provision of previsit forms alone for community centers serving low - income communities in which levels of formal education are low . future research is required to determine what strategies would be necessary to improve benefits from providing a previsit form : providing more extensive initial orientation to completing such forms ; making available staff or volunteers to provide ongoing assistance with completing the form such as what was provided in this intervention ; or offering a simpler form that focused on eliciting the patient 's main overall concerns and questions . a key question continues to be how best to both elicit patients ' key priorities and concerns for primary care visits and also provide necessary information to primary care providers to meet the medical needs of patients with chronic conditions . to meet the goal of achieving high quality , patient - centered primary care in all settings throughout the americas , it is critically important to continue developing and testing innovative and scalable interventions to better prepare both patients and their physicians to make the best use of the limited time in primary care visits [ 30 , 31 ] . our study findings support the hypothesis that providing active assistance to help patients identify their key questions and concerns before a clinic visit improves their visit satisfaction and belief that their objectives for the visit were met .
objectives . as in many settings , patients in community health centers in ecuador do not complete previsit forms or receive assistance to identify questions and concerns they would like to address in brief clinic visits with physicians . we examined the comparative effectiveness of providing ( 1 ) a previsit form to complete ; ( 2 ) a previsit form along with assistance in completing the form ; and ( 3 ) usual care . methods . parallel , three - arm randomized controlled trial in two health centers serving indigent to low - income communities in quito , ecuador , among 199 adult patients who took medications for at least one chronic condition . outcome measures were self - reported satisfaction with the visit , confidence in asking questions , and extent to which patients ' objectives were met . results . patients who received assistance in completing a previsit form were more than twice as likely as participants in usual care to report achieving everything they wanted during their visit ( aor 2.2 , p = 0.039 ) . there were no differences in any outcomes between the groups who received the previsit form with no assistance and usual care . conclusions . for high - quality patient - centered primary care , it is important to develop and test innovative and scalable interventions for patients and physicians to make the best use of limited clinic time .
You are an expert at summarizing long articles. Proceed to summarize the following text: this was a cross- sectional descriptive study performed on the high school and college students aged 16 - 18 . the statistical population consisted of 16 - 18 year old boy and girl students in tehran . the descriptive research is used to determine the sample size morgan table , that for population size 1000 and higher , we into consideration 384 . 422 samples have been used in this study . among the 19 education zones of tehran , five zones of north , south , east , west , and center were selected using the cluster random sampling method . in the second stage , a list of all public and private schools was provided , and high school students of these five zones were selected . then of each zone , 4 schools ( 2 boys and 2 girl s schools ) were selected with the simple random sampling method . next , 21 students with ages of 16,17 and 18 were selected from each school by the simple random sampling method . individually , with regard to secrecy and preservation of the moral values in the research the area of humanities , 422 questionnaires ( 211 boys and 211 girls ) were completed in order to assess the prevalence of borderline personality symptoms with a questionnaire for collecting adolescents demographic information with clinical interview by a trained clinical psychologist . revised diagnostic interview for borderlines ( dib - r ) the revised diagnostic interview for borderlines questionnaire ( dib - r ) , which is a semi - structured interview was used in this study . this questionare collects information in four domains that is thought to be of importance in the diagnosis of borderline personality disorder including : affect , cognition , impulse action patterns and interpersonal relationships . this assessment consists of 97 questions related to thinking , feeling and patient s behavior in the past two years . the interview is divided into 24 sub - sections , and information that is obtained from 22 sub - sections and is used for calibration of 22 scales shown with capitalized letters called the summary statements . each of these statements is an important diagnostic criterion for borderline personality disorder , and is used to assess the existence or nonexistence of this disorder . information of the other two sub - sections is used the inverse in opposite diagnosis of borderline disorder ( questions # 24 and 58 ) , and are used , respectively , in determining the patient s final score in parts of affect and cognition ( 12 ) . the overall has been ( kappa coefficient 0/70 and correlation coefficient of internal 0/80 ) . in some studies , the test , retest reliability has been good ( kappa = 0/71 ; r=0/71 ) . in a study , high internal reliability for the total score dib has been ( kuder - richardson = 0/75 ) ( 13 ) . the primary reliability , with the test - retest method on 30 participants within a week with the correlation coefficient ( dib - r ) was 0/81(14 ) . the psychometric characteristics of the ( dib - r ) spanish version were assessed in a sample of 156 patients with the possible diagnosis of borderline personality disorder . there were 29 men and 127 women with a mean age of 27.6 years ( standard deviation : 6/5 ; range : 18 - 45 ) . the spanish adaptation of the structured clinical interview for dsm - iii - r personality disorders ( scid - ii ) was used as gold standard . the dib - r showed good total internal consistency ( cronbach s alpha : 0/89 ) and high inter - rater reliability ( within - class correlation : 0/94 ) . using logistic regression analyses , the best cut - off was judged to be 6 or more , obtaining high sensitivity ( 0/81 ) , specificity ( 0/94 ) and moderate convergent validity of the diagnosis with the scid - ii ( kappa : 0/59)(15 ) . this was a cross- sectional descriptive study performed on the high school and college students aged 16 - 18 . the statistical population consisted of 16 - 18 year old boy and girl students in tehran . the descriptive research is used to determine the sample size morgan table , that for population size 1000 and higher , we into consideration 384 . 422 samples have been used in this study . among the 19 education zones of tehran , five zones of north , south , east , west , and center were selected using the cluster random sampling method . in the second stage , a list of all public and private schools was provided , and high school students of these five zones were selected . then of each zone , 4 schools ( 2 boys and 2 girl s schools ) were selected with the simple random sampling method . next , 21 students with ages of 16,17 and 18 were selected from each school by the simple random sampling method . individually , with regard to secrecy and preservation of the moral values in the research the area of humanities , 422 questionnaires ( 211 boys and 211 girls ) were completed in order to assess the prevalence of borderline personality symptoms with a questionnaire for collecting adolescents demographic information with clinical interview by a trained clinical psychologist . revised diagnostic interview for borderlines ( dib - r ) the revised diagnostic interview for borderlines questionnaire ( dib - r ) , which is a semi - structured interview was used in this study . this questionare collects information in four domains that is thought to be of importance in the diagnosis of borderline personality disorder including : affect , cognition , impulse action patterns and interpersonal relationships . this assessment consists of 97 questions related to thinking , feeling and patient s behavior in the past two years . the interview is divided into 24 sub - sections , and information that is obtained from 22 sub - sections and is used for calibration of 22 scales shown with capitalized letters called the summary statements . each of these statements is an important diagnostic criterion for borderline personality disorder , and is used to assess the existence or nonexistence of this disorder . information of the other two sub - sections is used the inverse in opposite diagnosis of borderline disorder ( questions # 24 and 58 ) , and are used , respectively , in determining the patient s final score in parts of affect and cognition ( 12 ) . the overall has been ( kappa coefficient 0/70 and correlation coefficient of internal 0/80 ) . in some studies , the test , retest reliability has been good ( kappa = 0/71 ; r=0/71 ) . in a study , high internal reliability for the total score dib has been ( kuder - richardson = 0/75 ) ( 13 ) . the primary reliability , with the test - retest method on 30 participants within a week with the correlation coefficient ( dib - r ) was 0/81(14 ) . the psychometric characteristics of the ( dib - r ) spanish version were assessed in a sample of 156 patients with the possible diagnosis of borderline personality disorder . there were 29 men and 127 women with a mean age of 27.6 years ( standard deviation : 6/5 ; range : 18 - 45 ) . the spanish adaptation of the structured clinical interview for dsm - iii - r personality disorders ( scid - ii ) was used as gold standard . the dib - r showed good total internal consistency ( cronbach s alpha : 0/89 ) and high inter - rater reliability ( within - class correlation : 0/94 ) . using logistic regression analyses , the best cut - off was judged to be 6 or more , obtaining high sensitivity ( 0/81 ) , specificity ( 0/94 ) and moderate convergent validity of the diagnosis with the scid - ii ( kappa : 0/59)(15 ) . the mean age in this study was 16/95 , and the standard deviation was 0/811 . the sample demographic data are presented in table 1 , the prevalence of borderline personality symptoms is shown in tables 1 , 2 , 3 , and statistical significance is demonstrated in table 4 . the present study ; the prevalence of borderline personality symptoms in the general population 16 - 18 years old , was 0/9 ; and with the results of other studies on children and adolescents are in agreement . ( 2001 ) on the over 1300 french high school students between the ages of 11 - 17 , that was 15% samples male and 17/2% samples female , from the screening personality disorders test and the revised diagnostic interview for borderlines ( dib - r ) was used . 2002 ) in a clinical comparing that on adolescents hospitalized ( 53% ) with adults ( 47% ) , they found that there was not a significant difference in the prevalence of bpd ( 17 ) . ( 1989 ) found that bpd is the most common personality disorder ( i.e. , 30% ) in a sample of depressed adolescents ( 9 ) . ( 1990 ) conducted a longitudinal assessment on the personality disorders of cluster a , b and c , particularly bpd , in children aged 13 , 16 , and 18 and found that those diagnosed in the cluster b , were 13 year olds ; 40% of the samples showed dysfunction personality , compatible is with personality disorders obtained from cluster b , the group age 18 years old . when the stability of bpd about 2 years in adolescents was studied , rate was relatively low stability ( for example , the range of 23% - 33%)(18 ) . the large epidemiological study on an english population was done by zanarini ( 2003 ) ; in which 3/3 % out of 10000 persons being 11 years old had full diagnostic criteria for bpd . figures found the fig 2% estimated prevalence in the adult general population is near and most of it ( apa , 2000 ) ( 19 ) . in a study the relationship between psychopathology in childhood and personality disorders in adulthood was investigated in a sample of 551 adolescents aged 9 - 16 . participants according to the dsm - iii - r criteria were assessed in three cases over 10 years . the results showed that diagnostic and prediction of personality disorder in adolescents in the same cluster of adults personality disorder is diagnosed independent of the axis i. moreover , the comorbidity of axis i and axis ii disorders was a significant amount more than of total adolescents personality disorder . the results obtained very similar for cluster b personality disorders in a period of 8 years in a community sample of adolescents show that diagnostic personality disorders , such as bpd may be in period adolescence not only is the value , also may help to inform for treatment and preventing the chain of maladaptive behaviors ; that is growing and become converting to intrinsic behavior and rooted ( 20 ) . in a study that leung sw , leung f ( 2009 ) did on 4110 high school students , the prevalence of bpd , with attention to accurate diagnostic methods used in this study was 2% among chinese adolescents in hong kong ( 21 ) . several researches showed that women are significantly more likely to have the criteria of bpd as compared to men , even when controlling for depression . the prevalence of bpd in female adolescents with conduct disorder and psychiatric in patient samples about 31% to 61% , while their male counterparts had a range of 0% to 39% ( 2225 ) this study showed bpd diagnostic criteria 11% with gender difference non - significant ( 10 ) . in a study done by carla sharp et al ( 2011 ) studied on 111 adolescents 12 to 17 years old with using of semi - structured clinical interviews , it showed that 23% of samples have bpd criteria ( 26 ) . in the present study the prevalence of borderline personality symptoms in boys 0/45 % of total and in girls 0/45 % of total . the association between demographic variables ( age , sex , location , parents occupation , parents kinship , parents education and birth order ) with borderline personality symptoms , only parents kinship with borderline personality symptoms showed a weak correlation ; that showed genetic is important in the incidence and prevalence sickness , particularly psychiatric disorders . generally , it seems that although the prevalence of borderline personality symptoms in this study , with many of studies done in this field is same orientation , but with some of study of the prevalence is different . first , this difference is can relate to cultural issues , religious and social norms in adolescents answer to questions , and second , this study done on non - clinical and most studies that have reported a high prevalence have done their research on clinical groups . results obtained from this study and other studies show that borderline personality symptoms from period adolescence ( 16 years old ) is visible and diagnosis ; and in answer to the original question this study , that , do borderline personality symptoms in adolescents is similar borderline personality disorder symptoms in adults ? answer is positive ; that may be behavioral science specialists can a separate category , such as conduct disorder for adolescents borderline personality symptoms to make in the dsm . this study showed 0/9% prevalence of borderline personality symptoms in high school students in tehran . although the findings of this study can not be generalized to the whole of iran , it can be used as a basis to further epidemiological studies for borderline personality symptoms in adolescents . also it can be used in planning specialists and country health planners , improvement , and health provide health services to adolescents .
objectivethis study aimed to assess the prevalence of borderline personality symptoms in 16 - 18 year old adolescents.methodsin this cross sectional - descriptive study , 422 high school students ( 211 boys , 211 girls ) aged 16 - 18 were selected by cluster random sampling and simple random sampling in 2011 - 2012 . the participants were assessed using the revised diagnostic interview for borderline questionnaire ( dib - r ) and demographic questionnaire . data were analyzed using pearson correlation coefficient and spearman correlation coefficient.resultsof the participants , 0/9% ( 0/22 % of the 16 year olds , 0.45 % of the 17 year olds and 0/22% of the 18 year olds ) were diagnosed with borderline personality symptoms . also , the prevalence of borderline personality symptoms in boys was 0/45 % of the total sample and it was 0/45 % of the total sample in girls . with respect to the relationship between demographic variables ( age , sex , location , parents occupation , parents kinship , parents education and birth order ) and borderline personality symptoms , only parents kinship showed a weak correlation with borderline personality symptoms.conclusionin the view of the prevalence of 0.9% of the borderline personality symptoms in adolescents , attention should be paid to the diagnosis and treatment of this disorder . furthermore , works need to be done to improve the mental health and quality of life of adolescents .
You are an expert at summarizing long articles. Proceed to summarize the following text: blepharokeratoconjunctivitis is a condition that is defined clinically as the presence of eyelid involvement by lid margin scarring , blepharitis , meibomitis , and chalazion or hordeolum , accompanied by epithelial or stromal keratitis , corneal scarring , vascularization , and thinning or perforation.1 it is a common condition in children , with the mean age at presentation around 10 years.1 children with blepharokeratoconjunctivitis often present with symptoms of eye redness ( 68.6%),1 photophobia ( 63.6%),2 tearing ( 50.0%),2 pain ( 21.6%),1 and irritation ( 13.7%).1 common clinical signs include corneal scarring ( 92.2% ) , superficial corneal vascularization ( 90.2% ) , meibomitis ( 86.3% ) , blepharitis ( 76.5% ) , punctate epithelial erosions or superficial punctate keratitis ( 74.5% ) , conjunctival hyperemia ( 51.0% ) , and chalazion ( 45.1%).1 blepharokeratoconjunctivitis has a chronic course characterized by exacerbation and remissions . recurrent and prolonged inflammation may lead to severe permanent ocular surface damage . the treatment for pediatric blepharokeratoconjunctivitis includes eyelid hygiene , warm compresses , and a combination of topical and systemic antibiotics.3 during acute exacerbations , a topical corticosteroid has been used to control conjunctival and corneal inflammation.2 blepharokeratoconjunctivitis is the result of an underlying cytokine and inflammatory - mediated process affecting both the meibomian glands and the ocular surface.4 topical corticosteroids inhibit proinflammatory cytokines by inhibiting transcriptase factors ( eg , nuclear factor-b ) . however , their long - term use is known to cause side effects such as reactivation of infectious keratitis , glaucoma , and cataracts . cyclosporin is an established immune modulating drug that was originally used to prevent graft rejection . topical preparation became commercially available in recent years for usage in ocular surface - related diseases.5 cyclosporin is a cyclic polypeptide produced as a metabolite by the fungus tolypocladium inflatum.6 it inhibits the activity of transcriptase factors of the nuclear factor of activated t cells , interfering with the induction of cytokines and other inducible genes required for the immune response.6 fukushima et al demonstrated in an animal model that immune - mediated blepharoconjunctivitis is t cell - mediated,7 and staphylococcus toxins may be the causative agent in humans.8 besides that , cyclosporin also showed better meibomian gland secretion and stable tear film compared to a conventional corticosteroid.4 cyclosporin has been reported to be helpful as a primary or adjunctive therapy in the treatment of dry eye,9 thygeson s superficial punctate keratitis,10 superior limbic keratoconjunctivitis,11 and meibomian gland dysfunction.12 here , a case of acute exacerbation of vision threatening bilateral blepharokeratoconjunctivitis in a young girl is reported . she did not respond to intensive treatment and topical corticosteroids ; however , responded well to topical cyclosporin without any side effects . a 9-year - old malay girl was diagnosed to have bilateral blepharokeratoconjunctivitis with intermittent exacerbation and was under the authors care for the past year . she was being treated with regular lid scrubbing using diluted baby shampoo , fusidic acid gel , and topical dexamethasone 0.1% as well as an intermittent course of oral doxycycline 50 mg daily for 46 weeks . her symptoms usually resolved after each course of intensive treatment , but the condition tended to exacerbate frequently . she developed acute onset of bilateral eye redness associated with poor vision in her right eye . the presence of corneal vascularization obscured the visual axis in the right eye , with old central corneal scarring ( figure 1 ) . in the left eye , there was also presence of peripheral corneal vascularization with old central corneal scarring ( figure 1 ) . the lid margin showed features of meibomian gland dysfunction with a presence of posterior lid margin telangiectasia , inflammation , and capping of the meibomian glands in both eyes . staphylococcal aureus was isolated from her conjunctival and lid swab , which was sensitive to fusidic acid . she was treated with intensive lid hygiene using diluted baby shampoo , fusidic acid gel every 8 hours , and topical dexamethasone 0.1% every 6 hours for both eyes . her right eye condition remained the same despite intensive treatments . a trial with topical cyclosporin a 0.5% every 6 hours was decided on , which was the authors first experience using topical cyclosporin for blepharokeratoconjunctivitis . there was a dramatic regression of corneal vascularization in both eyes after 3 days on topical cyclosporin ( figure 2 ) . after 1 week of treatment , both eyes showed white conjunctiva with resolved corneal vascularization ( figure 3 ) . her tear film also seemed to be more stable with improved tear break - up time . at 1-month follow - up , her ocular surface looked stable and healthy . the frequency of topical cyclosporin was reduced from every 6 hours to every 12 hours ; however , after 2 weeks on topical cyclosporin a 0.5% every 12 hours , there was a reappearance of conjunctival hyperemia and corneal vascularization . in view of this recurrence , it was decided that the frequency of topical cyclosporin a 0.5% every 6 hours would be maintained for a longer period . every attempt to taper cyclosporin treatment resulted in a reappearance of symptoms and signs of blepharokeratoconjunctivitis . it was decided that topical cyclosporin a 0.5% every 6 hours would be kept for up to 6 months before attempting to taper down the frequency . during this period , there was no ocular burning , eye stinging , nor conjunctival hyperemia noted during follow - up . follow - up at 6 months after cessation of treatment showed that there was no recurrence of the disease and the ocular surface was stable and healthy . treatment for blepharokeratoconjunctivitis is the same as treatment for posterior blepharitis or meibomian gland dysfunction,13 which includes eyelid hygiene , warm compresses , and a combination of topical and systemic antibiotics.3 daily lid hygiene removes debris and encrustations along the lid margin using cotton wool - tipped swab sticks with diluted baby shampoo , warm water , diluted bicarbonate solution,2 or special eyelid cleansers.3 in the current case report , the patient was being treated with regular lid scrubbing using diluted baby shampoo . common organisms isolated from conjunctival or lid swabs in patient with blepharokeratoconjunctivitis are s. aureus,2,3,14 s. epidermidis,2,3 and propionibacterium acnes.14 a variety of topical antibiotics has been used to reduce the bacterial load , including chloramphenicol,3 fusidic acid gel,1,3 levofloxacin,1 tobramycin,1 erythromycin,3 ciprofloxacin,3 and gentamicin.3 systemic antibiotics are often necessary to control the lid disease . oral erythromycin is an effective systemic antibiotic and tetracycline is contraindicated in children younger than 8 years because of its effect on dental enamel.3 in a study done by teo et al , children younger than 9 years of age were treated with oral erythromycin or augmentin , whereas older children were treated with oral doxycycline.1 the patient in the current case report was treated with an intermittent course of oral doxycycline because she is in the older age group . although corticosteroids are widely used as a primary ocular immunosuppressive agent , long - term use can induce various vision threatening side effects including glaucoma , cataracts , ulcers , and reactivation of viral keratitis.15 in the current patient , topical cyclosporin was introduced after an adequate trial of intensive treatments including steroids , which failed to show any improvement in the patient s condition . there was dramatic regression of the ocular surface inflammation with improved vision after initiation of topical cyclosporin . cyclosporin specifically interferes with the underlying mechanism in meibomian gland dysfunction by targeting t cells.6 cyclosporin has advantages over corticosteroids , since there is no suppression of normal host immune response and enhancement of collagenolytic enzyme production with subsequent corneal thinning . a study by rubin and rao showed that patients treated with cyclosporin for 12 weeks had increased meibomian gland secretion , a stable tear film , and a high mean secretion quality , with improvements in symptoms of blurred vision , itching , burning , lid erythema , and telangiectasia.4 ocular burning is the most common adverse event associated with the ophthalmic use of cyclosporin.9 no drug - related adverse events occurred in the patient in the current case report . based on this single case , results are promising but a full investigation with a large population would be required to change conventional clinical practice in terms of using cyclosporin as a first - line treatment to prevent permanent ocular surface damage in blepharokeratoconjunctivitis . this is a single case in which cyclosporin improved the status of the ocular surface . a large cohort study is required to justify its effectiveness in treating blepharokeratoconjunctivitis and to test its potential as an alternative immunosuppressive agent in comparison to conventional corticosteroids .
here , a case of vision threatening blepharokeratoconjunctivitis that responded well to topical cyclosporin is reported . a 9-year - old malay girl with a history of bilateral blepharokeratoconjunctivitis was regularly treated with lid scrubbing using diluted baby shampoo , fusidic acid gel , and topical steroids as well as an intermittent course of oral doxycycline for the past year . she developed acute onset bilateral eye redness associated with poor vision in her right eye . both eyes showed marked diffuse hyperemic conjunctiva with corneal vascularization . the presence of corneal vascularization obscured the visual axis in the right eye . the condition did not improve with regular intensive lid hygiene using diluted baby shampoo , fusidic acid gel , and topical steroids . she was started on topical cyclosporin a 0.5% every 6 hours . there was a dramatic regression of corneal vascularization after 3 days on topical cyclosporin , with marked improvement in visual acuity . this is a single case in which cyclosporin improved the status of the ocular surface . a large cohort study is required to justify its effectiveness in treating blepharokeratoconjunctivitis and to test its potential as an alternative immunosuppressive agent in comparison to conventional corticosteroids .
You are an expert at summarizing long articles. Proceed to summarize the following text: activity within neuronal networks is contained between the extremes of complete silence and exceeding neuronal discharges . this general statement may seem intuitively right but has severe and nontrivial consequences for the function of neuronal networks . several theoretical arguments and experimental findings support the notion that specific mechanisms secure a limited mean level of activity . information content within neuronal networks is maximal under conditions of sparse coding , which means that only a minority of all local neurons is activated above threshold . furthermore , neurons are severely damaged by both extremes , that is , prolonged inactivity [ 25 ] or severe hyperactivity during epileptic seizures . many different mechanisms contribute to regulation of overall neuronal activity , including intrinsic neuronal properties [ 7 , 8 ] and energy metabolism [ 9 , 10 ] . at the core of homeostasis , however , is the interplay between synaptic excitation and inhibition ( figure 1 ) . all neuronal circuits of higher animals contain excitatory and inhibitory transmitter systems forming intense feed - forward and feedback connections [ 11 , 12 ] . the functional architecture of such networks can already explain homeostatic regulation of activity to a certain degree , and excitatory feedback loops tend to build up activity , which is counterbalanced by dampening actions of inhibitory feedback connections . a further element of cortical and subcortical microcircuits is inhibition of inhibitory neurons , resulting in a net excitation of downstream target cells . this mechanism may serve further functions in synchronizing neuronal activity and can be mediated by specialized interneurons . in contrast , interactions between inhibitory neurons may also desynchronize neurons as , for example , renshaw cells in the spinal cord [ 14 , 15 ] . it should be noted that inhibitory neurons are not only important for balancing excitation . in several circuits , inhibitory neurons function as projection cells , rather than interneurons . for example , major projections within the basal ganglia and reticular nucleus of the thalamus and of the cerebellum are formed by gabaergic neurons [ 1618 ] . in such networks , activity - dependent modulation of inhibition may have specific effects beyond balancing excitation , for example , the generation of specific physiological or pathological oscillation pattern . homeostasis between excitation and inhibition can not be reduced to a simple rule of network wiring . recent evidence shows that inhibition has multiple specific functions within neuronal networks , far beyond a simple break [ 20 , 21 ] . moreover , inhibitory strength is not constant but must adapt to dynamically changing patterns and degrees of network activity . it does therefore not come as a surprise that recent work has elucidated multiple mechanisms of plasticity at inhibitory synapses [ 4 , 2224 ] . an important subset of these mechanisms mediates homeostatic plasticity , that is , adaptation of inhibitory efficacy to the overall activity within a local network . indeed , several lines of evidence suggest that gabaergic efficacy is upregulated in hyperactive networks [ 2529 ] and downregulated under conditions of reduced activity [ 5 , 30 , 31 ] . such homeostatic reactions can , in principle , be mediated by multiple pre- and postsynaptic mechanisms . a particularly important regulatory system , however , is the concentration of the main mammalian inhibitory transmitter gaba ( -aminobutyric acid ) . this paper shall summarize the molecular elements and functional mechanisms involved in regulation of gaba concentration within vesicles , cells , and in the extracellular space . we will quote experimental evidence indicating that gaba is homeostatically regulated during physiological and pathological changes of network activity . finally , we will consider how molecular determinants of gaba concentration can be targeted by drugs for pharmacological therapy of neurological or psychiatric diseases . in the mammalian cns , inhibition is mediated by the amino acids gaba and glycine . the gabaergic system has been intensely explored during recent years and will therefore be the main focus of this paper . as a starting point , we will briefly summarize the main functional and structural elements of gabaergic synapses . gaba - gated ion channels are selectively permeable for chloride and bicarbonate and have reversal potentials close to cl equilibrium ( ecl ) . these channels are mostly termed gabaa receptors , but a molecularly and pharmacologically distinguishable subset has also been termed gabac receptors until recently , as discussed by olsen and seighart . in most cases , the increase in chloride ( and bicarbonate ) conductance resulting from activation of ionotropic gaba receptors causes inhibition of the respective neuron , that is , decreased probability of action potential generation . this is easy to understand in cases where ecl is more negative than the membrane potential , such that opening of gabaa receptors causes hyperpolarisation and enhances the distance between membrane potential and action potential threshold . however , inhibition can also be mediated by more complex biophysical mechanisms , for example , shunting of the local membrane resistance , which can also counteract excitatory inputs . even depolarizing actions of gaba can , in certain cases , be inhibitory [ 3335 ] . conversely , excitatory actions of gaba may occur in specific situations , including early developmental stages [ 3639 ] and maladaptive processes , for example , in chronic epilepsy [ 40 , 41 ] . the occurrence of depolarizing gaba responses under physiological conditions is presently subject to some controversy [ 39 , 42 ] . gabab receptors , in contrast , are members of the family of g - protein - coupled proteins and react to gaba binding by dimerisation and activation of downstream signal cascades . these include decreased probability of transmitter release and increase in pre- and postsynaptic k conductance [ 45 , 46 ] . a complete survey of gabaergic mechanisms at the molecular , cellular , and network level is far beyond the scope of this paper . rather , we will highlight three principles of organization of gaba - mediated inhibition that are particularly important for understanding how gaba regulates network activity . one important mechanism is tonic inhibition , which results from diffusely distributed gaba within the extracellular space of networks , thereby reducing excitability of all local neurons ( figure 1 ) . recent evidence has shown that tonic inhibition is of major importance for reducing firing probability of defined types of neurons within cortical networks [ 24 , 4749 ] . in some cells , background levels of gaba in neuronal tissue have been estimated to reach high - nanomolar to low - micromolar concentrations [ 51 , 52 ] . in good accordance with this relatively low concentration , extrasynaptic gaba receptors have particularly high agonist affinity [ 47 , 53 , 54 ] . at the other extreme , phasic inhibition is mediated by locally and temporally restricted release of gaba from synaptic terminals . this action causes a short , exponentially rising and falling of the postsynaptic chloride conductance which can last from few to tens of milliseconds [ 50 , 55 ] . most gabaergic neurons seem to form such specific synaptic sites for phasic inhibition , but recent evidence indicates that there are also specialized interneurons which release gaba for tonic inhibition [ 5660 ] . tonic inhibition depends on special gaba receptors , which can be selectively modulated by drugs , for example , neurosteroids . these specific receptor isoforms may be important in the pathophysiology of depression and withdrawal symptoms . work on different networks has revealed an unprecedented multitude of different gabaergic neurons which are classified by their somatic location , dendritic branching , axonal projection , afferent synaptic integration , intrinsic membrane properties , and expression of molecular markers , especially neuromodulatory peptides and calcium - binding proteins . extensive classification systems have been established for different circuits , for example , for the rodent neocortex [ 63 , 64 ] and the hippocampus [ 13 , 65 ] . moreover , introducing the juxtacellular recording technique has enabled recordings from individual interneurons in behaving animals and subsequent in - depth structural analysis . these data have shown that different types of interneurons are specialized to organize different patterns of network activity . in accordance with the heterogeneity and functional specialization of different cell types , experiments and computer modelling have revealed important functions of inhibitory interneurons in networks beyond merely dampening excitation . interneurons turned out to play a key role in organizing the spatiotemporal activity of local networks , especially during synchronous network oscillations [ 6873 ] . complementary neuroanatomical work has highlighted the structural basis for this function : interneurons have highly divergent axonal projections , cell type - specific afferent and efferent connectivity , and synchronizing mutual connections . all these properties favour synchronous rhythmic inhibition of large populations of principal cells [ 13 , 69 , 71 , 7476 ] . it should be noted that the connections between excitatory projection cells and inhibitory interneurons provide an automatic homeostatic mechanisms at the network level . feed forward or feedback inhibition is driven by excitatory inputs or outputs , respectively , from remote or local excitatory neurons . this mechanism does automatically recruit inhibitory neurons in an activity - dependent manner and , hence , balance local activity ( figure 1 ) . the molecular organization of synapses is highly complex , and a complete review would be beyond the scope of this paper . we will restrict our remarks to some families of molecules that are crucial for understanding homeostatic regulation of gaba concentration ( figure 2 ) . like many other neurotransmitters , gaba acts on ionotropic as well as metabotropic ion channels . gabaa receptors are pentameric ion channels composed out of a large variety of 19 homologous subunits [ 32 , 77 , 78 ] . work during the past decades has elucidated numerous functional differences between molecular subtypes of gabaar , including different expression patterns , differential modulation by benzodiazepines , neurosteroids and zn , different compartmentalization within neurons , and different agonist affinity [ 32 , 54 , 79 ] . gabaars with low agonist affinity appear to be clustered at postsynaptic sites , whereas receptors with high affinity are mostly found extrasynaptically [ 47 , 48 ] . the underlying sorting mechanisms are partially known and involve specific subsynaptic sorting signals within the gamma subunit and interactions with postsynaptic scaffolding proteins like gephyrin and collybistin [ 8083 ] . extrasynaptic receptors , in contrast , are formed by subunits mediating high agonist affinity including 4 , 6 , and subunits [ 32 , 47 ] . this distinction reflects the different concentrations of gaba at both sites : whereas synaptically released gaba may reach transient concentrations of ~1.53 mm [ 84 , 85 ] , extrasynaptic transmitter concentration has been estimated to lie in the low micromolar range of about 0.22.5 m [ 47 , 51 , 52 , 86 ] . gabaergic auto- or heteroreceptors have been described at the axon terminals of various neurons , including spinal cord afferents , hippocampal mossy fibres , schaffer collaterals , cerebellar interneurons , and pituitary terminals . depending on the gaba - induced change in membrane potential and local membrane resistance , presynaptic gabaa receptors may increase or decrease transmitter release . gabab receptors , on the other hand , are g - protein - coupled transmembrane molecules which are activated by low concentrations of gaba and form dimers which then trigger secondary signalling cascades [ 4345 ] . at presynaptic terminals , activation of gababrs reduces gaba release , forming the typical negative feedback loop of autoreceptor - mediated synaptic gain control . gabab receptors are also present at glutamatergic terminals , pointing towards regular spillover of gaba from inhibitory to excitatory synapses ( figure 1 , [ 47 , 9395 ] ) . postsynaptically , gababrs hyperpolarize and inhibit neurons by activating inwardly rectifying kir channels , giving rise to the slow or late phase of inhibition that follows fast , gabaar - mediated effects and lasts several hundred milliseconds . furthermore , gabab receptors can also mediate tonic inhibition , exerting negative control on overall network activity . while gaba receptors act as detectors of local gaba concentration , the regulation of gaba itself is achieved by several specialized molecular mechanisms mediating transport , sequestration , synthesis , and the degradation of gaba . membrane - bound gaba transporters move gaba across the cell membrane ( figure 2 ) . the direction and efficacy of this na - coupled transport results from the driving electrochemical gradient and is directed inwardly in most situations [ 98 , 99 ] . however , upon strong depolarization or altered ion homeostasis , gaba transporters can also reverse direction . this mechanism leads to nonvesicular release of gaba which may be of special importance in pathophysiological situations [ 60 , 100 , 101 ] . gaba transporters appear in four different isoforms with affinities around 7 m for rat gat-1 , 8 m for rat gat-2 , 12 m for rat gat-3 , and 93 m for rat bgt-1 [ 102106 ] . terminology of gaba transporters is not fully compatible between rats and mice . in the following , we use the abbreviations for rat gaba transporters where ratgat-1 = mousegat1 ; ratgat-2 = mouse gat3 ; ratgat-3 = mousegat4 ; ratbgt-1 = mouse gat2 . gat-1 is the prevailing neuronal isoform in the rodent brain , and gat-3 is strongly expressed in glial cells [ 108110 ] . expression of different gat isoforms is , however , overlapping , so that selective modulation of one isoform will always affect more than one cell type . it might therefore turn out impossible to achieve a strictly selective block of glial or neuronal gaba uptake with conventional pharmacological tools . similar to gat-1/3 , there are membrane - bound glutamate transporter molecules at presynaptic terminals of inhibitory interneurons , namely eaac1 ( also called eaat3 ) [ 111113 ] . moreover , neurons can synthesize glutamate from glutamine which can also be taken up by specialized transporters ( see below ) [ 114 , 115 ] . gabaergic neurons express both mature isoforms of glutamate decarboxylase , gad65 and gad67 [ 116 , 117 ] , that convert the excitatory amino acid into gaba . the smaller isoform gad65 is directly associated to presynaptic vesicles , indicating that glutamate , once present in the presynaptic cytosol , can be rapidly used for vesicular enrichment of gaba . indeed , there are direct protein interactions between gad65 and the vesicular gaba transporter vgat (= viaat , vesicular inhibitory amino acid transporter ) , suggesting that conversion of glutamate into gaba and subsequent vesicular uptake of the transmitter may be strongly coupled processes . more recently the amino acid glutamine has long been known as the immediate precursor for glutamate . in the extracellular space , enrichment of glutamate in excitatory central neurons involves uptake through specific glutamate transporters by glia cells , conversion into glutamine , export via there is increasing evidence for a similar role of this glutamate / glutamine cycle in gaba synthesis . indeed , inhibitory interneurons in the hippocampus express the system a transporter snat1 , but not snat2 . recordings of epileptiform activity in rodent brain slices in vitro have revealed functional evidence for boosting of inhibition by glutamine via this mechanism [ 124127 ] . using high - resolution recordings of miniature ipscs in conjunction with pharmacological manipulation of glutamine levels and glutamine transport , these studies showed that glutamine can serve as a source for gaba , especially under conditions of increased synaptic activity . more recent evidence from rat hippocampal slices showed that the contribution of glutamine to vesicular gaba content is more pronounced in immature tissue , and that glutamine forms a constitutive source of vesicular gaba in immature hippocampal synapses on ca1 pyramidal cells . at later stages this loss of function for constitutive gaba release under resting conditions goes along with an age - dependent decline in expression of snat1 , both absolutely and in relation to the gaba - synthesizing enzyme gad65 . within presynaptic terminals of gabaergic neurons , gaba is enriched in vesicles by the vesicular inhibitory amino acid transporter ( vgat = viaat ) . this protein is embedded in the vesicular membrane and uses the electrochemical gradient for h to shuffle gaba into small synaptic vesicles [ 129133 ] . additionally , chloride gradients between vesicle lumen and presynaptic cytosol may contribute to the vesicular loading of gaba [ 129 , 131 ] . this is a prerequisite for the observed gabaergic / glycinergic cotransmission by single vesicles in the spinal cord . modelling studies and biochemical data suggest that vesicular gaba uptake may achieve an~1000-fold increase of the transmitter in vesicles as compared to the presynaptic cytosol . on the other side , recent evidence suggests that gabaergic synaptic vesicles are leaky , implying generation of a dynamic equilibrium between accumulation and loss of gaba , given that there is enough time to reach such a steady state [ 132 , 136 ] . taking this bidirectional transport into account , the leaky bathtub model of synaptic vesicles comes to rather low estimates of concentration gradients between cytosol and the inner vesicle space [ 132 , 135 , 137 ] . the reaction is catalysed by gaba transaminase ( gaba - t ) which is present in mitochondria of glial cells and neurons [ 138140 ] . it is estimated that more than 90% of all gaba in the mammalian cns is degraded in this way and contributes to energy metabolism in the tricarbonic acid cycle . in summary , there are several different molecular pathways and compartments for enrichment , synthesis , and degradation of gaba ( figure 2 ) . the resulting concentration of gaba in synaptic vesicles and in the extracellular space depends on the equilibrium between these mechanisms . it should be clearly stated that the absolute concentrations of gaba in the presynaptic cytosol , in vesicles , and in the extrasynaptic space are not known . the affinity constants of extrasynaptic gaba receptors may serve as a rough estimate of background concentrations ( 0.22.5 m ) . direct measurements from rat cerebrospinal fluid yielded similar or slightly higher values which may be lower in humans . the highly dynamic time course of transmitter concentration in the synaptic cleft , on the other hand , has been estimated based on experimental and theoretical work in different types of neurons . peak concentrations may be as high as 0.3 to 3 mm [ 85 , 142147 ] . the cytosolic gaba concentration is most difficult to estimate or measure , especially since most of the neuronal gaba pool is used for energy metabolism rather than for synaptic inhibition . it should be explicitly stated that none of the above - given numbers has been directly measured . indeed , our knowledge on local gaba concentrations in different compartments is far from sufficient . this is even more concerning when we take into account the enormous heterogeneity of neurons [ 20 , 63 , 65 ] , the different microarchitecture of different local circuits , and activity - dependent changes in gaba release and ionic homeostasis . a major challenge is the lack of quantitative data about key molecules and structures : how many gaba - uptake molecules are present at a given inhibitory synapse ? an important example for progress in this quantitative molecular approach to subcellular structure and function is the recent work on the vesicular proteasome by takamori and colleagues . different lines of evidence support the view that the cellular and molecular mechanisms mentioned above make important contributions to homeostatic synaptic plasticity . this term covers changes of intrinsic and synaptic neuronal properties , which maintain the mean network activity within a determined range [ 4 , 28 ] . taking into consideration that network states change rapidly with changes in vigilance and behavioural state [ 148 , 149 ] , individual neurons can change their activity at least by a factor of ~6 in different network patterns . nevertheless , under normal conditions , networks do neither fall into complete silence , nor into pathological hyperactivity . most circuits contain specialized inhibitory cells which are activated by external afferent excitatory inputs ( feedforward inhibition ) or by collaterals from efferent excitatory axons ( feedback inhibition ) as illustrated in figure 1 . these inhibitory control loops ensure that excitatory neurons are inhibited in an activity - dependent manner . it should be noted , however , that inhibitory interneurons are much more than a brake or recent evidence has revealed many other functions for these heterogeneous neurons : they are critical for organizing the complex spatial and temporal patterns of network oscillations [ 70 , 71 ] , selective gating of defined inputs or outputs , suppression of background activity , and precise timing of action potentials . corresponding with these specific functions , we are gaining increasing insight into the complexity of gabaergic signalling , diversity of interneurons , and plasticity of inhibitory synapses . notwithstanding these recent findings , however , inhibition does still have its traditional function , that is , limitation of neuronal activity . with respect to network homeostasis , this control function must adapt to changing degrees of activity in the local network . several lines of evidence indicate that modulating gaba content of inhibitory interneurons is a key mechanism in this regulation process . for example , repetitive hyperactivity in the hippocampus of chronically epileptic rats causes upregulation of gads , the key enzymes for production of gaba . conversely , the partial deafferentation of somatosensory cortex resulting from partial limb amputations leads to a downregulation of gaba , but not of gads [ 30 , 152 ] . these findings indicate that gaba levels are increased or decreased , respectively , in response to increasing or decreasing network activity . long - term changes in excitability , such as described above , require regulation of protein expression . multiple studies from excitatory synapses show that changes in synaptic activity do indeed include lasting effects on protein synthesis and synaptic protein content [ 153 , 154 ] . the underlying mechanisms involve calcium signalling in dendrites and nuclei [ 30 , 155 ] . it would be of special importance to understand the activity - dependent regulation of key proteins such as gad , vgat , and others . interestingly , bdnf ( brain - derived neurotrophic factor ) increases expression of gad , indicating that neurotrophins are involved in inhibitory homeostatic plasticity . this would be well compatible with the general role of these molecules in activity - dependent plasticity . surprisingly , genes for inhibitory transmission can also be upregulated in excitatory , glutamatergic neurons following periods of enhanced activity . this intriguing finding suggests that excitatory neurons can adopt an active role in synaptic inhibition in certain situations . such a dual phenotype has been clearly demonstrated in dentate granule cells , a major excitatory input cell type in the rodent hippocampus [ 157159 ] . the axons of granule cells , called mossy fibres , form strong glutamatergic synapses on proximal dendrites of ca3 pyramidal cells and do also contact inhibitory interneurons in this region ( an example of feedforward inhibition ) . upon strong repetitive stimulation or following epileptic seizures , mossy fibres start expressing proteins needed for the production and vesicular storage of gaba . electrophysiological measurements show that this gabaergic phenotype is indeed functional , giving rise to mixed excitatory and inhibitory potentials in ca3 pyramids . the gabaergic phenotype of mossy fibres seems to be more pronounced in the juvenile brain , consistent with the general principle of enhanced plasticity in immature neurons . while the dual phenotype of granule cells may be an extreme example , several observations indicate that similar activity - dependent changes in expression of gabaergic molecules affect the vesicular pool of gaba in typical inhibitory interneurons . for example , expression of vgat is altered following ischemia or excitotoxic stimulation [ 160162 ] . these changes go along with altered composition of the vesicular proteome , indicative of altered supply or release of gabaergic vesicles . at a shorter time scale , gaba levels might be regulated by activity - dependent uptake of transmitter molecules . experimental evidence for such changes came from direct injection of glutamate or glutamine [ 124 , 125 ] into hippocampal slices . both approaches increased the amplitudes of miniature inhibitory postsynaptic currents ( mipscs ) , indicating that the precursors had indeed been used to fuel the vesicular transmitter pool . consistent with these findings , blocking membrane - bound transporters for glutamine , gaba , or glutamate can reduce the size of ipscs [ 128 , 162 , 165 , 166 ] . the relative contribution of gaba , glutamate , or glutamine uptake to the vesicular gaba pool remains , however , unknown . it can be expected that the contribution of different transmitter transporters differs among neuronal subtypes , brain regions , and developmental stages [ 128 , 167 ] . however , due to the fast action of uptake molecules , it is well possible that homeostatic adaptations of intravesicular gaba concentration occur at time scales of few seconds . strong activation of axons in the ca1 area of mouse hippocampal slices results in a rapid increase of mipsc amplitudes , with onset time below 20 s. this increase is dependent on uptake of glutamate and gaba , indicating that increased extracellular transmitter concentrations in active neuronal networks automatically provide more fuel to the pool of releasable gaba , thereby constituting a negative feedback loop . we have already discussed that tonic activation of gaba receptors by ambient transmitter concentrations provides a major mechanism for regulation of excitability [ 47 , 48 , 50 ] . it may , therefore , well be that changes in gaba uptake , production , and release cause altered tonic inhibition , possibly mediated by specialized subtypes of interneurons . it is also unclear how much nonvesicular release of gaba by reverse transport contributes to ambient gaba concentration . situations of hyperactivity may favour such release mechanisms by sustained depolarization and altered local ion homeostasis [ 59 , 60 , 100 , 168 ] . enhancing gabaergic inhibition is useful for the treatment of several pathological situations , including chronic pain , sleep disorders , anxiety , and most importantly epilepsy . in accordance with the principles outlined above , one approach is blocking gaba degradation by gaba transaminase ( gaba - t ) , using the suicide inhibitor -vinyl - gaba ( gvg ) . indeed , this drug does increase gaba levels in the brain [ 169 , 170 ] and has anticonvulsant efficacy [ 171 , 172 ] . studies at the single cell level show that gvg increases miniature ipsc amplitude , consistent with a dynamic regulation of vesicular gaba concentration by the equilibrium between synthesis and degradation [ 173 , 174 ] . clinical use of gvg is , however , limited due to pathological changes of retinal cells and resulting scotoma . an alternative approach suited to enhance synaptic gaba levels is the redirection of gaba uptake from glia to neurons . in glial cells , in contrast , neuronal gaba uptake can recycle the amino acid for use as a transmitter . unfortunately , the molecular distinction between glial and neuronal gaba uptake is not strict , although there is some bias for gat-1 in neurons and gat-3 in glia [ 108110 ] . in summary , there is no doubt that changes in gaba concentration contribute significantly to network homeostasis in health and disease . more quantitative information about sources , compartmentalization , and local concentration of gaba is urgently needed , not at least in order to develop more specific drugs for reconstituting excitation - inhibition balance in pathological situations .
gabaergic inhibition is an important regulator of excitability in neuronal networks . in addition , inhibitory synaptic signals contribute crucially to the organization of spatiotemporal patterns of network activity , especially during coherent oscillations . in order to maintain stable network states , the release of gaba by interneurons must be plastic in timing and amount . this homeostatic regulation is achieved by several pre- and postsynaptic mechanisms and is triggered by various activity - dependent local signals such as excitatory input or ambient levels of neurotransmitters . here , we review findings on the availability of gaba for release at presynaptic terminals of interneurons . presynaptic gaba content seems to be an important determinant of inhibitory efficacy and can be differentially regulated by changing synthesis , transport , and degradation of gaba or related molecules . we will discuss the functional impact of such regulations on neuronal network patterns and , finally , point towards pharmacological approaches targeting these processes .
You are an expert at summarizing long articles. Proceed to summarize the following text: precocious puberty in boys is defined as the appearance of the initial signs of sexual maturation at an age that is more than 22.5 sd below the population mean , which is 9 yr for boys . precocious puberty can be gonadotropin dependent due to the early activation of pulsatile gonadotropin secretion or independent of gonadotropins , as is the case with testotoxicosis or leydig cell tumours . the aetiology of an early activation of pulsatile gonadotropin secretion may be secondary to an organic lesion within the central nervous system ( hydrocephaly , tumours , meningitis , etc . ) or due to activating mutations of the genes encoding kisspeptin or its receptor gpr54 . however , in many cases the cause that triggers the onset of puberty can not be identified . gonadotropin - dependent precocious puberty is more prevalent in girls ; nevertheless , the proportion of cases of precocious puberty secondary to organic lesions is higher in boys . gonadotropin - releasing hormone analogues ( gnrha ) is the treatment of choice for gonadotropin - dependent precocious puberty of central origin . therapy induces a reversible pituitary desensitization of gnrh receptors , thus suppressing the production of gonadotropins and subsequently that of sex steroids . slowing the advancement of bone age is the primary goal of gnrha therapy in order to improve final height [ 1 , 2 ] . monitoring treatment effectiveness during gnrha therapy resides in multiple items . growth velocity , secondary sexual characteristics , and bone maturation are the main clinical parameters . serum lh and fsh measured by ultrasensitive assays are used for the assessment of pituitary desensitization [ 5 , 6 ] , and serum testosterone levels are used to monitor testicular response to treatment . measurement of testicular volume is clinically utilized for the assessment of pubertal development in boys ; the increase in testicular volume beyond 4 ml is typically used to define the onset of puberty , either normal or precocious . the initial increase in testicular volume is due to sertoli cell proliferation induced by fsh . subsequently , testosterone provokes sertoli cell maturation characterized by sertoli cell proliferation arrest and triggers pubertal spermatogenesis in the seminiferous tubules , which mainly accounts for the dramatic enlargement of testis size to 1525 ml . other early signs of pubertal sertoli cell maturation , which occur even before serum testosterone rises , are the changes in serum levels of two specific sertoli cell products : the decrease in anti - mllerian hormone ( amh ) [ 911 ] and the increase in inhibin b [ 1214 ] . in the present study , our primary objective was to assess functional changes in sertoli cells , based on the measurement of serum amh and inhibin b during treatment with gnrha and after its withdrawal in boys with central precocious puberty . boys diagnosed with central precocious puberty at the division of endocrinology of the hospital de nios ricardo gutirrez , a tertiary paediatric public hospital in the city of buenos aires , argentina , and treated with gnrha between 1995 and 2013 were included in the study . patients were excluded if history chart or results of serum hormone determinations ( fsh , lh , testosterone , and amh ) were not complete or available . a clinical examination was performed at each visit to assess pubertal stage according to marshall and tanner and to determine testicular volume by comparison with prader 's orchidometer . lh , fsh , testosterone , inhibin b , and amh serum concentrations were assessed periodically before , throughout , and after treatment with gnrha . the need for informed consent was waivered owing to the observational design of the study , in which most of the procedures followed the standard care of patients with central precocious puberty , and because results of serum amh and inhibin b levels were not considered for decision making . amh was determined using an ultrasensitive enzyme linked immunoassay specific for human amh ( immunotech , beckman - coulter co. , marseilles , france ) , following the manufacturer 's instructions as previously published . the analytical sensitivity of the assay , defined as the lowest amh concentration significantly different from the calibrator zero , was 2.3 intra- and interassay coefficients of variation were , respectively , 10.5% and 9.4% for a serum amh concentration of 700 serum inhibin b was measured using two - site enzyme - linked immunosorbent assays ( oxford bio - innovation ltd , oxon , uk ) as previously described . recombinant inhibin b ( genentech , san francisco , ca , usa ) was used as standard . serum fsh and lh were measured by time - resolved immunofluorometric assays ( ifma , delfia ; perkinelmer , inc . by wallac oy , turku , finland ) . iu / l , according to the 2nd who is 80/552 for lh and irp 94/632 for fsh , respectively . intra- and interassay coefficients of variation for ifma method were 3.2 and 7.3% for lh and 2.3 and 5.2% for fsh , as previously published . hormone concentrations below the limit of the assay detection were assigned a concentration equivalent to the minimum detectable value in the respective assay . multiple regression analysis was performed to assess the association between serum inhibin b levels and testis volume during followup after gnrha treatment . statistical analyses were performed using graphpad prism version 5.01 for windows ( graphpad software , san diego , ca , usa ) and ibm spss statistics ( ibm corporation , somers , ny , usa ) . six boys with central precocious puberty were included in the study ( table 1 ) . their median age at diagnosis was 3 years ( range 0.85.5 ) with a bone age of 8.5 years ( range 1.2510.5 ) . in three cases , precocious puberty was due to a hypothalamic hamartoma and in the other three it was idiopathic . patients were treated with triptorelin acetate every 28 days at a 110190 g / kg dose . patient 5 was still in treatment at the moment of the study . at diagnosis , testicular volume was between 2 and 9 ml ( figure 1(a ) ) , fsh was 1.75 iu / l ( range 0.615.36 ) ( figure 1(b ) ) , median basal lh was 3.75 iu / l ( range 0.8811.20 ) ( figure 1(c ) ) , and testosterone was 419 ng / dl ( range 12878 ) ( figure 1(d ) ) . median peak lh after an acute gnrh test was 35.30 iu / l ( range 6.9084.90 ) ( table 1 ) . serum amh was low in the four patients older than 1 year ( median 67 pmol / l , range 50165 ) but was within the normal range in the two patients aged 1 year ( 645 and 475 pmol / l ) ( figure 1(e ) ) . interestingly , in patient 5 , who was diagnosed at 12 months of age , amh ( 475 pmol / l ) was within the normal prepubertal range . due to social difficulties gnrha treatment could not be started until the age of 2.2 yr when serum amh had decreased to 131 pmol / l . before treatment serum inhibin b was elevated ( 400 pg / ml , testis volume decreased but remained moderately above the normal prepubertal size in most patients ( figure 1(a ) ) . as expected , serum gonadotropins and testosterone showed a progressive decrement toward normal prepubertal levels and remained so until gnrha withdrawal , when they increased again to pubertal levels ( figures 1(b)1(d ) ) . amh increased progressively and remained within the normal prepubertal range during the whole treatment ( figure 1(e ) ) . after gnrha withdrawal , amh progressively declined concomitantly with the rise in serum testosterone , as in normal puberty [ 16 , 18 ] . serum inhibin b levels decreased moderately throughout gnrha therapy attaining its nadir after 24 months of treatment . thereafter , they remained in the upper half of normal prepubertal age range ( figure 1(f ) ) . a multiple regression analysis using patient and testis volume as the independent variables and inhibin b levels as the dependent variable showed a significant positive correlation between testicular volume and serum inhibin b levels during treatment ( r = 0.561 p < 0.001 ) ( figure 2 ) . interestingly , during gnrha treatment the lowest levels of inhibin b were observed in patients 1 , 2 , and 6 , in whom testicular volume achieved normal prepubertal range , whereas inhibin b remained higher in patients 3 and 4 , who maintained testicular volume above 4 ml in spite of suppressed fsh levels . after treatment withdrawal , inhibin b rose toward normal pubertal levels concomitantly with the reactivation of the gonadotropin axis . by means of serum amh and inhibin b assessment , this work depicts the functional changes occurring in the seminiferous tubule compartment and especially in sertoli cells in response to gnrha treatment in boys with central precocious puberty . our results show that the abnormally early maturation process suffered by sertoli cells in boys with precocious puberty in response to testosterone is reversible when patients are efficaciously treated with gnrha , most probably reflecting the decrease to prepubertal levels in intratesticular testosterone concentration . on the contrary , inhibin b levels do not always decrease to prepubertal values although fsh secretion is also curtailed by gnrh administration , most probably indicating that the increased mass of sertoli cells is not fully reverted and continues secreting inhibin b independently of fsh stimulation . most of our patients had low serum amh levels for their chronological age when central precocious puberty was diagnosed , showing the well - known inhibition exerted by androgens on sertoli cell amh production . the inhibitory action of androgens predominates over the stimulatory effect of fsh on sertoli cell amh expression [ 19 , 20 ] . conversely , fsh action on sertoli cells is reflected by the initial increase in testicular volume and serum inhibin b. amh downregulation and inhibin b upregulation also reflect the entry into meiosis of germ cells . in fact , the onset of meiosis is associated with a further decline in amh expression , independently of testosterone , during pubertal development . concerning inhibin b production by the testis , the immature sertoli cell synthesizes the /b dimer , whereas the pubertal sertoli cell essentially produces the subunit under fsh control ; the b subunit expression is mainly regulated by local factors produced by meiotic germ cells . during gnrha treatment amh attained normal prepubertal levels , as previously described , which accounts for the usefulness of sertoli cell serum markers to monitor the efficacy of gnrha treatment in order to produce a sufficient decrease in gonadotropin production so as to lower intratesticular androgen action to prepubertal levels . the functional maturation of sertoli cells induced at pubertal onset , reflected in amh downregulation by androgens , seems to be a completely reversible process when treated soon before its establishment , as was the case in patients 14 of our series and in previously published cases . on the contrary , sertoli cells do not revert to a fully immature state if the action of intratesticular androgens has persisted for years or decades , as shown in adult males receiving gnrha treatment for prostate cancer . a noteworthy observation in our study was that serum amh remained in the normal prepubertal range in patients with central precocious puberty diagnosed within the first year of life , suggesting that the elevated androgen levels were unable to inhibit amh production in young infants . interestingly , testicular volume also remained within the prepubertal range in these patients , probably indicating that pubertal spermatogenesis had not been triggered . these observations are in line with the recent description that sertoli cells are physiologically insensitive to the direct action of androgens in the first year after birth because they do not express the androgen receptor until later in life [ 2426 ] . concordantly , in our patient 5 , amh was at prepubertal levels when diagnosed at 12 months of age and , because treatment could not be immediately installed , serum amh decreased to pubertal levels at age of 2.2 years , supporting the concept of a gradual increase in androgen receptor expression within the sertoli cell population . at diagnosis , serum inhibin b levels were elevated in all patients , resembling what occurs under normal pubertal development at tanner stages ii to iii . treatment with gnrha resulted in a reduction in serum inhibin b levels in all patients . however , the magnitude of this decrement was variable amongst patients . in some patients , inhibin b remained in the upper normal range during treatment , in correlation with the persistence of moderately elevated testis volume , despite suppressed fsh secretion . this probably reflects that the increased mass of sertoli cells induced by elevated fsh until diagnosis is not fully restored to the prepubertal stage and continues secreting inhibin b , independently of fsh stimulation . we believe that serum levels of inhibin b reflects the addition of two pools : a basal one related to the mass effect of the sertoli cell population independent of the gonadotropins and a second one related to the stimulation exerted by fsh . the mass effect is evidenced by the observation that testicular volume remains over the normal prepubertal volume in a subset of patients under gnrha treatment , concomitantly with higher serum inhibin b levels . it could be argued that these elevated serum inhibin b levels result from an insufficient inhibition of the gonadotropin axis with gnrha treatment . this seems unlikely , however , since serum levels of lh and testosterone were efficaciously suppressed and resulted in a normalization of serum amh . in summary , we describe the different functional changes observed in sertoli cells of boys with central precocious puberty according to their age at the moment of diagnosis . sertoli cell markers provide additional tools to diagnose central precocious puberty in boys older than 1 year and to monitor gnrha treatment efficacy . the observation that serum amh does not decrease in patients below 1 year confirms the lack of androgen receptor expression in sertoli cells in early infancy . finally , we hypothesize that serum levels of inhibin b represent the addition of two pools , one related to the stimulation exerted by gonadotropins and another one to the mass effect of the sertoli cell population independent of the gonadotropins .
we aimed to describe the functional changes of sertoli cells , based on the measurement of serum anti - mllerian hormone ( amh ) and inhibin b during treatment with gnrha and after its withdrawal in boys with central precocious puberty . six boys aged 0.8 to 5.5 yr were included . amh was low at diagnosis in patients > 1 yr but within the normal range in younger patients . amh increased to normal prepubertal levels during treatment . after gnrha withdrawal , amh declined concomitantly with the rise in serum testosterone . at diagnosis , inhibin b was elevated and decreased throughout therapy , remaining in the upper normal prepubertal range . in patients with testicular volume above 4 ml amh remained higher in spite of suppressed fsh . after treatment withdrawal , inhibin b rose towards normal pubertal levels . in conclusion , amh did not decrease in patients < 1 yr reflecting the lack of androgen receptor expression in sertoli cells in early infancy . serum inhibin b might result from the contribution of two sources : the mass of sertoli cells and the stimulation exerted by fsh . sertoli cell markers might provide additional tools for the diagnosis and treatment followup of boys with central precocious puberty .
You are an expert at summarizing long articles. Proceed to summarize the following text: recently , a great advance was achieved in the understanding of pathogenesis of autoimmune diabetes , particularly regarding the molecular mechanism of pancreatic -cell apoptosis and immunological significance of -cell apoptosis . hence , it has been shown that pancreatic -cell death is important not only in the final effector phase of autoimmune type 1 diabetes ( 1,2 ) but also in the initiation of -cell autoimmunity ( 3,4 ) . such progress was mostly achieved by employing murine models of autoimmune diabetes because application of most essential genetic manipulation or immunological intervention is possible only in animal models . murine autoimmune diabetes models and human type 1 diabetes have many common characteristics such as the presence of insulitis , requirement for specific mhc haplotypes and autoimmune responses to autoantigens including glutamic acid decarboxylase ( gad ) . thus , many recent progress obtained employing murine autoimmune diabetes models would be applicable to human type 1 diabetes , implying important therapeutic potential . autoreactive t lymphocytes are the most important effector cells in murine autoimmune diabetes ( 5 - 7 ) , and probably in human type 1 diabetes . they will ultimately induce apoptosis of -islet cells in murine autoimmune diabetes ( 8,9 ) , while apoptosis of pancreatic -cells in human type 1 diabetes has not been demonstrated because of the difficulty in procuring human pancreatic tissue and inability to synchronize disease process in human type 1 diabetes . we have shown that ifn/tnf synergism is responsible for murine pancreatic -cell apoptosis both in vitro and in vivo ( 2 ) , which was critically dependent on stat1 activation by ifn ( 1 ) . in vivo role of stat1 activation by ifn other death effector molecules such as fas ligand ( fasl ) might be an effector molecule for a small number of autoreactive lymphocytes ( 10 ) but not for the majority of final death effector cells ( 9,11 ) . ifn appears to sensitize otherwise resistant pancreatic islet cells or insulinoma cells to tnf -mediated apoptosis by activating stat1/irf-1 signal pathway ( 2 ) . most such pancreatic -cell death and signal pathways were studied in murine autoimmune diabetes models . such findings might also be relevant in human type 1 diabetes also . in an effort to elucidate the possible mechanism of human pancreatic islet cell apoptosis leading to human type 1 diabetes , we investigated if similar cytokine synergism could induce apoptosis of human pancreatic islet cells and similar signal molecules are induced in them . human pancreatic islets were obtained from brain - dead patients between 1998 and 2002 at samsung medical center , using a modification of the automated method for human islet isolation ( 12 ) . briefly , 350 ml of hank 's buffered salt solution ( hbss ) containing 9.1mol / l collagenase solution ( liberase , boehringer - mannheim , mannheim , germany ) was injected through the pancreatic duct after cannulation . the pancreas was loaded into a stainless steel digestion chamber , and islets were separated during a continuous digestion process for 30~45 min . during the recirculation phase ( flow rate , 85 ml / min ) , intrachamber temperature was increased at a rate of 2/min by the passage of the solution through a stainless steel coil immersed in a water bath ( 50 ) . the chamber containing the distended pancreas was gently agitated and samples were taken every 2 min to monitor digestion . after 20~30 min of recirculation , digestion was stopped by dilution in 400 ml / min ice - cold hbss . in this phase , the digested tissue was rapidly collected in sterile bottles containing 200 ml fcs . purification of the islets was carried out over discontinuous euro - ficoll gradients ( 1.108 , 1.096 , 1.037 and hbss ) . purified islets were then washed twice , evaluated for purity , and counted after dithizone ( sigma , st . the pellet was resuspended in 3 ml warm 53.7mol / l trypsin-3 mmol / l edta . after incubation at 37 for 5 min and pipetting for 2~3 min , islets became invisible . then , cells were washed with warm rpmi-10% fcs at 1,500 rpm for 5 min . single islet cells were frozen in liquid nitrogen until use . at the time of assays , single islet cells were thawed and 1.410 cells were plated per single well of 96-well plates . viability of the islet cells after thawing was above 90% , as judged by trypan blue staining and acridine orange / propidium iodide staining . isolated human pancreatic islet cells were grown in dmem containing 15% fbs , 2 mm glutamine , and penicillin - streptomycin ( gibco - brl , gaithersburg , md ) . recombinant human tnf and recombinant human il-1 were purchased from r&d systems ( minneapolis , mn ) . louis , mo ) unless stated otherwise . cells ( 210/well for human single islet cells ) were seeded in 96-well microtiter plates and treated with various combinations of cytokines for the indicated time periods . the optimal concentrations of cytokines for the cytotoxic action were 1,000 u / ml for ifn , 10 ng / ml for tnf , and 17.5 ng / ml for il-1. after cytokine treatment , the medium was removed and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ( mtt , 0.5 mg / ml ) was added followed by incubation at 37 for 2 hr in co2 incubator . after a brief centrifugation , supernatant was carefully removed and dmso was added . after insoluble crystals were completely dissolved , absorbance at 540 nm was measured using thermomax microplate reader ( molecular devices , sunny vale , ca ) . morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 2.5g / ml of dna - binding bisbenzimide hoe33342 fluorochrome ( calbiochem , san diego , ca ) , followed by an examination on a fluorescence microscope . for transmission electron microscopy , cells were fixed in 4% glutaraldehyde/1% paraformaldehyde/0.2 m phosphate ( ph 7.2 ) at 4 for 2 hr . after two washes in 0.2 m phosphate , the cell pellets were postfixed with 2% oso4 in the same buffer for 30 min . the pellets were dehydrated in ethanol and then in 100% propylene oxide , followed by embedding at 37 overnight and 60 for another 3 days . ultrafine sections were cut and examined on an electron microscope ( hitachi h7100 , 75 kv ) . cells were lysed in triple - detergent lysis buffer ( 50 mm tris - cl , ph 8.0 , 150 mm nacl , 0.02% sodium azide , 0.1% sds , 1% np-40 , 0.5% sodium deoxycholate , 1 mm pmsf ) . an equal amount of protein for each sample was separated by 10% or 12% sds - page and transferred to hybond ecl nitrocellulose membrane ( ge healthcare life sciences , buckinghamshire , uk ) . after blocking with 5% skim milk , the membranes were sequentially incubated with one of the primary antibodies ( rabbit anti - mouse irf-1 , santa cruz ; rabbit anti - phospho - stat1 , new england biolabs , ipswich , ma ) and then hrp - conjugated secondary antibodies ( anti - rabbit igg , ge healthcare life sciences , buckinghamshire , uk ) , followed by ecl detection ( ge healthcare life sciences , buckinghamshire , uk ) . human pancreatic islets were obtained from brain - dead patients between 1998 and 2002 at samsung medical center , using a modification of the automated method for human islet isolation ( 12 ) . briefly , 350 ml of hank 's buffered salt solution ( hbss ) containing 9.1mol / l collagenase solution ( liberase , boehringer - mannheim , mannheim , germany ) was injected through the pancreatic duct after cannulation . the pancreas was loaded into a stainless steel digestion chamber , and islets were separated during a continuous digestion process for 30~45 min . during the recirculation phase ( flow rate , 85 ml / min ) , intrachamber temperature was increased at a rate of 2/min by the passage of the solution through a stainless steel coil immersed in a water bath ( 50 ) . the chamber containing the distended pancreas was gently agitated and samples were taken every 2 min to monitor digestion . after 20~30 min of recirculation , digestion was stopped by dilution in 400 ml / min ice - cold hbss . in this phase , the digested tissue was rapidly collected in sterile bottles containing 200 ml fcs . purification of the islets was carried out over discontinuous euro - ficoll gradients ( 1.108 , 1.096 , 1.037 and hbss ) . purified islets were then washed twice , evaluated for purity , and counted after dithizone ( sigma , st . the pellet was resuspended in 3 ml warm 53.7mol / l trypsin-3 mmol / l edta . after incubation at 37 for 5 min and pipetting for 2~3 min , islets became invisible . then , cells were washed with warm rpmi-10% fcs at 1,500 rpm for 5 min . single islet cells were frozen in liquid nitrogen until use . at the time of assays , single islet cells were thawed and 1.410 cells were plated per single well of 96-well plates . viability of the islet cells after thawing was above 90% , as judged by trypan blue staining and acridine orange / propidium iodide staining . isolated human pancreatic islet cells were grown in dmem containing 15% fbs , 2 mm glutamine , and penicillin - streptomycin ( gibco - brl , gaithersburg , md ) . recombinant human tnf and recombinant human il-1 were purchased from r&d systems ( minneapolis , mn ) . cells ( 210/well for human single islet cells ) were seeded in 96-well microtiter plates and treated with various combinations of cytokines for the indicated time periods . the optimal concentrations of cytokines for the cytotoxic action were 1,000 u / ml for ifn , 10 ng / ml for tnf , and 17.5 ng / ml for il-1. after cytokine treatment , the medium was removed and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ( mtt , 0.5 mg / ml ) was added followed by incubation at 37 for 2 hr in co2 incubator . after a brief centrifugation , supernatant was carefully removed and dmso was added . after insoluble crystals were completely dissolved , absorbance at 540 nm was measured using thermomax microplate reader ( molecular devices , sunny vale , ca ) . morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 2.5g / ml of dna - binding bisbenzimide hoe33342 fluorochrome ( calbiochem , san diego , ca ) , followed by an examination on a fluorescence microscope . for transmission electron microscopy , cells were fixed in 4% glutaraldehyde/1% paraformaldehyde/0.2 m phosphate ( ph 7.2 ) at 4 for 2 hr . after two washes in 0.2 m phosphate , the cell pellets were postfixed with 2% oso4 in the same buffer for 30 min . the pellets were dehydrated in ethanol and then in 100% propylene oxide , followed by embedding at 37 overnight and 60 for another 3 days . ultrafine sections were cut and examined on an electron microscope ( hitachi h7100 , 75 kv ) . cells were lysed in triple - detergent lysis buffer ( 50 mm tris - cl , ph 8.0 , 150 mm nacl , 0.02% sodium azide , 0.1% sds , 1% np-40 , 0.5% sodium deoxycholate , 1 mm pmsf ) . an equal amount of protein for each sample was separated by 10% or 12% sds - page and transferred to hybond ecl nitrocellulose membrane ( ge healthcare life sciences , buckinghamshire , uk ) . after blocking with 5% skim milk , the membranes were sequentially incubated with one of the primary antibodies ( rabbit anti - mouse irf-1 , santa cruz ; rabbit anti - phospho - stat1 , new england biolabs , ipswich , ma ) and then hrp - conjugated secondary antibodies ( anti - rabbit igg , ge healthcare life sciences , buckinghamshire , uk ) , followed by ecl detection ( ge healthcare life sciences , buckinghamshire , uk ) . treatment of single human islet cells with cytokines disclosed that combination of ifn , tnf and il-1 induced significant death after 5 days of incubation . when the effect of individual cytokine was studied , a combination of ifn and tnf induced death of human islet cells while single cytokine did not exert significant cytotoxicity on human islet cells ( fig . the addition of il-1 to the ifn/tnf combination had only a minor effect on islet cell viability . our results showing death of human islet cells by the triple combination of ifn , tnf and il-1 is similar to previous reports by others employing human islet cells ( 13 - 16 ) . percent death of human islet cells by the cytokine combination in this investigation was similar to a previous report ( 13 ) , and was smaller than that of murine islet cells by the similar cytokine combination even after prolonged incubation for 5 days ( 2 ) . most of the death of human islet cells by the triple combination could be explained by ifn/tnf combination because the addition of il-1 to the ifn/tnf combination increased human islet cell death only to a small degree , consistent with a previous paper ( 13 ) . hoechst 33342 staining and electron microscopic examination showed that the death of human islet cells by ifn/tnf combination was a typical apoptosis characterized by nuclear condensation and fragmentation with preserved plasma membrane integrity ( fig . these results are similar to previous reports showing nuclear condensation or tunel nuclei after cytokine treatment of human islet cells ( 15,16 ) . next we asked if ifn activates stat1 and induces irf-1 which has been reported to be important for induction of tnf susceptibility in otherwise resistant murine islet cells or cancer cells ( 2,17 ) . immunoblot analysis using antibody to phosphorylated stat1 showed that stat1 became activated 30 min after ifn treatment , similar to murine insulinoma cells ( 2 ) ( fig . irf-1 was also induced by ifn treatment of human islet cells for 48 hrs again similar to murine insulinoma cells suggesting that stat1 activation and irf-1 induction by ifn may sensitize human islet cells to tnf-induced apoptosis , similar to the case of murine islet cell death by ifn/tnf synergism ( 2 ) ( fig . these results were similar to our previous study using murine islet / insulinoma cells and suggests the possibility that intracellular signal activated by ifn may sensitize otherwise resistant human pancreatic islet cells to tnf-induced apoptosis , which could not be proved because of shortage of human islet cells , the absence of immortalized human islet cells and unavailability of genetic manipulation system . a recent paper has shown that bim , a bh3-only proapoptotic bcl-2 family member , is induced in human islets or rat islet cells after treatment with ifn+tnf which plays an important role in -cell death by ifn+tnf synergism ( 18 ) . in that model , bim induction was attributed to ifn-induced activation of stat1 which was bound to bim promoter ( 18 ) . while detailed biochemical and cellular mechanism of human islet cell death by cytokines could not be investigated further because of limited availability of human islets for diabetes research , our results suggest that the death of human islet cells is basically similar to that of murine islet cells .
fasl , perforin , tnf , il-1 and no have been considered as effector molecule(s ) leading to -cell death in autoimmune diabetes . however , the real culprit(s ) of -cell destruction have long been elusive despite intense investigation . previously we have suggested ifn/tnf synergism as the final effector molecules in autoimmune diabetes of nod mice . a combination of ifn and tnf but neither cytokine alone , induced classical caspase - dependent apoptosis in murine insulinoma and pancreatic islet cells . ifn treatment conferred susceptibility to tnf-induced apoptosis on otherwise resistant murine insulinoma cells by stat1 activation followed by irf-1 induction . here we report that ifn/tnf synergism induces apoptosis of human pancreatic islet cells . we also observed stat1 activation followed by irf-1 induction by ifn treatment in human islet cells . taken together , we suggest that ifn/tnf synergism could be involved in human islet cell death in type 1 diabetes , similar to murine type 1 diabetes .
You are an expert at summarizing long articles. Proceed to summarize the following text: gallbladder torsion is a rare condition requiring urgent cholecystectomy for a gangrenous change of the gallbladder . elderly women are most commonly affected with this condition as the number of reports has been recently increasing with aging population.12 anomalous attachment of the gallbladder to the liver with long or absent mesentery is the most common cause of gallbladder torsion . the most common anatomical variants include a floating gallbladder with a long and short mesentery containing cystic artery and cystic duct.3 however , a different mechanism may underlie gallbladder torsion . the present study reports of a rare type of gallbladder torsion , in which the fundus of the gallbladder was rotated counterclockwise along the axis near the end of mesenteric attachment of gallbladder to the liver . a strong peristaltic movement of the colon may precipitate the torsion of gallbladder fundus , which is not firmly attached to the liver . only four cases of such gallbladder torsion , which underwent surgery , have been reported in the literature.4567 this study presents a case in which fundus rotate along the axis of gallbladder . an 87-year - old woman complaining of abdominal pain in the right upper quadrant , nausea and vomiting for 4 days was admitted to the emergency room . she was moderately dehydrated with fever ( 38.5 ) but her vital signs were normal . on physical examination , a large , firm and tender mass was palpable on the right abdomen . liver function test was normal . computed tomography of abdomen showed a large and small cavity ( fig . 1a ) . the large cavity with the largest diameter of 15 cm had wall thickening and contained a large stone . the 3 cm - sized small cavity under the liver seemed to be a portion of normal gallbladder . the wall of large cavity was thickened and seemed to be the gangrenous portion distal to the twisted body of gallbladder . ultrasonography demonstrated a portion of gallbladder rotating in a counterclockwise direction along the axis of gallbladder . doppler ultrasonography confirmed blood flow in the small cavity proximal to the twisted portion , but there was no blood flow in the huge cavity distal to the twisted portion of the gallbladder ( fig . , a normal cystic duct , gallbladder neck , and proximal body was found to be attached firmly on the gallbladder bed . moreover , the distal portion of gallbladder was twisted 360 degree in counterclockwise direction around the axis of gallbladder resulting in huge distension with severe gangrenous change of distal gallbladder ( fig . histopathology showed mild inflammation found in the proximal part of the gallbladder in contrast to necrosis and gangrenous changes found in the distal gallbladder . although , gallbladder torsion may occur in all ages , it is an elderly disease . age - related weight loss and visceroptosis may be etiologically related to gallbladder torsion.2 female patients are more commonly affected than male , with the female to male ratio of 3 : 1.1 once complete torsion occurs , the patient suffers from acute upper right quadrant pain that mimics acute cholecystitis . due to the lack of specific symptoms and signs , it is rarely diagnosed preoperatively based on clinical findings . however , recently , with the advances of imaging diagnosis , several reports of preoperative diagnosis of gallbladder torsion have been published . the lack of blood flow by the duplex ultrasonography was helpful in the diagnosis.8 marano et al.3 described three anatomic variants of gallbladder attachment to liver responsible for gallbladder torsion . the first variant is a " free - floating gallbladder " suspended only by the mesentery of cystic duct . the second variant is elongated , narrow - based and freely mobile mesentery and rotation occurs along the axis of the narrow mesentery . an exceedingly rare third variant consists of a normally fixed gallbladder to a mobile liver lobe free of its coronary and triangular ligaments.9 in addition , to these mechanisms , as shown in our case , twisting of fundus along the axis of gallbladder body is another mechanism of gallbladder torsion . age - related laxity of gallbladder tissue may be a factor of the elongated fundus which rotated in a counterclockwise direction along the axis of gallbladder . a strong peristaltic movement of the colon may precipitate the counterclockwise torsion of elongated gallbladder fundus . so far , very few cases of such gallbladder torsion , which underwent surgery , have been reported in the literature.456 only four cases of gallbladder torsion caused by this mechanism have been reported so far ( table 1 ) . one case of the reports showed two gallbladder cavities , which were separated by the twisted portion of gallbladder on the ultrasonography , as shown in our case . in this case , normal looking proximal gallbladder that was shown in abdominal computed tomography led to confusion but ultrasonography detected the twisted portion , and this made it possible to trace the portion of twist in an anticlockwise direction . furthermore , doppler ultrasonography confirmed impairment of blood flow in the distal cavity whereas it demonstrated normal blood flow in the proximal cavity . microscopic findings of the distal gallbladder wall showed infraction in the background of chronic cholecystitis compared to the relatively normal wall of proximal gallbladder . some authors reported similar cases with intermittent torsion of the fundus , which were relieved by endoscopic retrograde cholangiopancreatography . based on the gross and microscopic morphology , the study concluded that the patient had normal attachment of the gallbladder mesentery to the liver , but the elongated mobile fundus caused by age - related laxity of the gallbladder tissue and peristaltic movement of the colon precipitated the rotation of the fundus . her accompanying gallbladder stone of 3 cm in size might be another precipitating factor for the torsion .
abnormal attachment of the gallbladder to the liver is the main cause for gallbladder torsion . however , the present study reports a rare case of gallbladder torsion in which a portion of fundus is rotated along the axis of body . so far , very few similar cases have been reported . an 87-year - old woman complaining right upper quadrant abdominal pain for 4 days was admitted . her body temperature was 38.5 with moderate dehydration . a large tender mass was palpated on the right abdomen extending to the right iliac fossa . computed tomography of abdomen showed a large cavity with a diameter of 15 cm containing a big stone and a small normal looking gallbladder . ultrasonographic scan showed a twisted portion of the gallbladder torsion . during emergency laparotomy , the middle portion of the gallbladder was found to be twisted counterclockwise with huge gangrenous gallbladder distal . the proximal body of the gallbladder was spared and attached firmly to the liver . cholecystectomy was performed and the patient was discharged 2 weeks later without postoperative complications . histological findings of specimen were consistent with operative findings . the current study reports on a rare case of gallbladder torsion by reviewing previous studies .
You are an expert at summarizing long articles. Proceed to summarize the following text: meteoric ablation is the source of metals such as calcium , sodium , and iron in the mesosphere and lower thermosphere ( mlt ) [ plane et al . , these metals occur as neutral atoms in layers between 80 and 100 km and atomic ions which become dominant above 95 km . lidar observations of the neutral metal layers have shown that ca is significantly depleted with respect to na and fe : the ca / na and ca / fe column abundance ratios are ~0.01 and 0.004 [ plane et al . , 2015 ] , compared to their chondritic ratios ( in ci type carbonaceous chondrites ) of 1.05 and 0.069 , respectively [ asplund et al . , 2009 ] . the most likely reason for this depletion is differential ablation , where the highly refractory ca only ablates from meteoroids which reach temperatures above 2200 k , compared to the relatively volatile elements na and fe which largely ablate below 2000 k [ carrillosanchez et al . , however , in contrast to the neutral ratios , the observed atmospheric ca / na and ca / fe ratios are ~0.4 and 0.03 [ kopp , 1997 ] , which are much closer to their respective ci ratios . this result implies that there are important differences in the ionmolecule chemistries of these metals . in this paper we focus on calcium and iron chemistries , because ca and fe are chemically reactive ions and therefore behave more alike , compared with the chemically inert na ion . in the mlt , these ions are largely produced by charge transfer reactions with no and , to a lesser extent , o2 ( photoionization is a much less important ionization process [ plane et al . , 2015 ] ) . for example [ rutherford et al . , 1972 ] , ( r1)ca+no+ca++no above 110 km , the slow dielectronic / radiative recombination of the metallic ions with electrons becomes the dominant neutralization pathway [ bones et al . , 2016 ] . however , between 90 and 100 km the dominant pathway is reaction with o3 to produce the metal oxide ion [ broadley et al . , 2007 ] , ( r2)ca++o3cao++o2followed by dissociative recombination ( dr ) with electrons ( r3)cao++eca+o since dr reactions generally have rate coefficients > 10 cm molecule s [ florescumitchell and mitchell , 2006 ] , if reactions r2 and r3 were the only chemistry occurring , the lifetime of ca would be ~10 s , with consequently much lower concentrations than measured by lidar [ gerding et al . , 2000 ; granier et al . , 1985 ; raizada et al . , 2011 ] and rocketborne mass spectrometry [ kopp , 1997 ] . in fact , the reaction of cao with atomic o ( r4)cao++oca++o2converts the metal oxide ion back to the atomic ion , thus slowing the overall rate of neutralization [ broadley and plane , 2010 ] . the rate coefficients k 1 [ rutherford et al . , 1972 ] , k 2 [ broadley et al . , 2007 ] , and k 4 [ broadley and plane , 2010 ] have been measured previously , as have the rate coefficients for the analogous reactions of fe [ rollason and plane , 1998 ; rutherford and vroom , 1972 ; woodcock et al . , we have recently reported the first measurement of the dr of feo [ bones et al . , 2016 ] . the present paper describes a study of reaction r3 , whose rate coefficient does not appear to have been reported previously . we then use the measured value of k 3 to understand why the ca / fe ratio between 90 and 100 km is nearly an order of magnitude larger than the ca / fe ratio . finally , we examine the role of ionmolecule chemistry in the phenomenon of sporadic ca layers ( cas ) . these are very thin layers of ca which appear suddenly , generally at altitudes around 95 km , and can have peak concentrations more than an order of magnitude above the background average ca atom layer [ gerding et al . , simultaneous commonvolume measurements of ca and ca by lidar provide a unique tool for determining whether cas are produced by the neutralization of ca in descending sporadic e layers , as proposed originally to explain sporadic na layer formation from na [ cox and plane , 1998 ] . the measurements were made in a flowing afterglow system with a pulsed laser source of metallic ions . the system is described in detail elsewhere [ bones et al . , 2016 ] . briefly , the system consists of a stainless steel flow tube connected to a differentially pumped quadrupole mass spectrometer ( hiden hpr60 ) running in positive ion mode with pulse counting . an ar / electron plasma is generated by flowing he through a microwave discharge cavity ( mccarroll cavity , 145 w at 2450 mhz ) and then introducing a flow of ar into the helium / electron plasma , whereupon the charge is transferred to the ar largely through penning ionization . the electron density was measured 12.5 cm upstream of the target by a langmuir probe ( hiden espion ) . all experiments were conducted at pressures of 1 torr and a temperature of 295 k. cao ions ( and neutral cao molecules ) were introduced into this plasma by the pulsed laser ablation of a solid target . a neodymium : yttrium / aluminum / garnet laser ( 8 mj pulse at 532 nm , 10 hz repetition rate ) was directed at a sample of ca metal with a heavily oxidized surface , mounted on a rotating rod . isotopic analyses of the ablated ions showed negligible contamination of the signal by fe ( which is isobaric with cao at m / z = 56 ) . for some experimental runs , the laser pulse was loosely focused on the target to change the ratio of cao to cao generated in the ablation pulse . the relative concentration of cao was measured by the mass spectrometer as a function of flow rate ( 35 slm ) , which is inversely proportional to the time available for the cao ions to react with the electrons in the plasma before they enter the mass spectrometer ( 2.13.5 ms , corresponding to flow velocities of 69113 m s ) . the skimmer cone between the flow tube and the first stage of the mass spectrometer had a 4.0 mm orifice biased at 19 v , and the skimmer cone between the first and second stages of the mass spectrometer had a 1.8 mm orifice biased at 89 v. each cao measurement was the sum of the m / z = 56 signal from three repeat accumulations of 500 laser shots each . these repeat measurements had an average standard deviation of 15% , although for measurements at low flow velocities the standard deviation could be as high as 30% . during the course of an experiment , over typically 23 h , the cao ablation efficiency varied slowly ( usually decreasing ) , and this was corrected for by taking reference cao signals periodically ( at a median flow velocity of 91 m s ) and fitting a normalization function . materials : he ( 99.995% , boc gases ) was purified by passing through a trap of dry molecular sieve ( 4 , 12 mm , alfa aesar ) cooled to 77 k. ar ( 99.9999% , boc gases ) was used without further purification . the calcium shot ( sigmaaldrich ) was at least a decade old and extensively oxidized . the ablation of the oxidized ca target produced pulses of cao and cao ( figure s1 in the supporting information ) . the cao then undergoes charge transfer with ar ions in the plasma : ( r5)cao+ar+cao++ar the upper limit for the rate coefficient k 5 can be calculated from modified langevin theory [ su and chesnavich , 1982 ; troe , 1985 ] to be 8.2 10 cm molecule s , using a theoretical dipole moment of 8.8 d and polarizability of 6.6 10 cm for cao [ broadley and plane , 2010 ] . the interactions of cao , cao , ar , and e in the flow tube are then described by the following coupled differential equations : ( e1)dcao+dt = k5ar+caokdiffcao++k3ecao+ ( e2)dcaodt=kdiffcao+k5ar+cao ( e3)dar+dt=kdiffar++k5caoar+ ( e4)dedt=kdiffe+k3cao+e the average electron diffusion rate k diff was 948 96 s at 1.0 torr . this was determined by measuring the absolute electron density with the langmuir probe and varying the flow velocity at constant pressure ( figure s2 ) . the electron density immediately after formation of the ar plasma was then estimated to be ( 3.2 0.6 ) 10 cm . in the diffusion rate of ar , k diff , the dominant positive charge carrier was assumed to match the electron diffusion rate under the conditions of ambipolar diffusion which apply because the electrons and ions rapidly equilibrate to the bath gas temperature at the relatively high total pressure of 1 torr [ smirnov , 2001 ] ) . k diff was estimated to be 3.7% smaller than k diff in the bath gas where the ar / he ratio was 1:10 [ bones et al . , the diffusion coefficients of neutral cao are calculated ( using the method described in self and plane ) to be 464 and 95 torr cm s in he and ar , respectively , which corresponds to a wall loss rate k diff of 651 s. relations e1e4 were solved numerically to predict the relative cao concentration at the downstream end of the flow tube . there are two unknowns : k 3 and the initial neutral cao concentration ( [ cao]0 ) . each data set was then normalized to the cao produced by pulsed ablation at the fastest flow velocity , in order to remove the daytoday variation in the ablation efficiency from the ca target . the data were then averaged in six reaction time bins with an average bin width of 1.4 10 s , and a neldermead iterative simplex algorithm [ nelder and mead , 1965 ] used to find the best fit of the kinetic model to the six experimental points , by varying k 3 and [ cao]0 . the fitting was weighted to the number of data in each time bin , which varied between 10 and 32 . note that in order to model the timeresolved cao concentration , the model actually requires the product k 5 [ cao ] in relations e1 and e2 ; since we fix k 5 to the calculated langevin limit ( see above ) , [ cao]0 is then the other unknown fitted parameter . figure 1a is a contour plot of as a function of k 3 and [ cao]0 , where ( e5)2=i=1ncaoexpt , i+caomodel , i+caoexpt , i+2 ( a ) plot of the surface as a function of k 3 and initial neutral cao concentration produced by pulsed ablation of the ca target . the white cross indicates the position of the minimum , and the white contour the 1 envelope of uncertainty . ( b ) abundance of cao as a function of time in the flow tube between the ablation source and detection at the mass spectrometer : measurements ( solid circles with 1 error bars ) , model fit ( solid line ) , and model run with the upper and lower limits of k 3 ( dashed lines ) . the minimum on the surface yields k 3(295 k ) = ( 3.0 1.0 ) 10 cm molecule s , where the uncertainty estimate is discussed below . the corresponding value for [ cao]0 is 1.9 10 molecule cm . the ratio of [ cao]0/[cao]0 produced directly by ablation is estimated to be 17 5 . this ratio decreased by 45% when the ablation laser was loosely focused , as expected because a greater fraction of ions would have been produced . the uncertainty in k 3 was estimated by using monte carlo selection of the theoretically calculated and experimentally measured parameters used in the kinetic model , within their uncertainties listed in table 1 ( assuming a tophat probability distribution ) . the model was then rerun to compare with the measurements : if the simplex algorithm found a minimum in the surface , and the resulting value of k 3 was within the range of 1 101 10 cm molecule s expected for a dr reaction [ florescumitchell and mitchell , 2006 ] , then the value of k 3 was accepted . the mean and standard deviation of k 3 from 2500 model fits then provided the final value of k 3 and the estimate of its uncertainty . although the determination of k 3 is sensitive to the flow velocity and electron diffusional wall loss , these parameters are measured with high precision . in contrast , k 3 is much less sensitive to the estimated [ cao]0/[cao]0 ratio and estimate of k 5 using langevin theory , but these parameters have a greater assigned uncertainty ( table 1 ) and so make a similar contribution to the overall uncertainty of k 3 ( see the supporting information for further details ) . the white contour line in figure 1a depicts the envelope of uncertainty around the minimum , which is 44% below the contour line . figure 1b shows a plot of the measured abundance of cao ( with 1 standard deviation ) as a function of reaction time . the dashed lines show the model output with the upper and lower bounds of k 3 . note that the overall uncertainty is larger than the uncertainty of the measurements because the monte carlo analysis included additional uncertainties ( table 1 ) when determining the uncertainty of k 3 . in order to study the ionmolecule chemistry of calcium in the mlt , we assign a t temperature dependence to k 3 , which is typical for dr reactions [ florescumitchell and mitchell , 2006 ] . reaction r3 is 45% slower than the dr reaction of feo at room temperature [ bones et al . , we now examine the consequences of this for explaining the very different ca / fe and ca / fe ratios in the 90100 km region of the atmosphere . the dashed black line shows the [ ca]/[fe ] ratio profile measured by rocketborne mass spectrometry . the mean and standard deviation were calculated from seven flights covering a range of latitudes and local times ( note that the geometric , rather than arithmetic , mean is used because of the small data set ; the geometric mean is defined as x=x1x2xnn ) . the dashed blue line shows the mean [ ca]/[fe ] ratio computed from simultaneous and common volume lidar measurements of ca and fe at khlungsborn , germany ( 54n ) . data from 22 nights ( each with 29 h of measurements ) were used ; the total data set is ~120 h of measurements . the measurements were performed between april 1997 and march 1999 . vertical profiles ( 90100 km ) of the measured ca / fe ratio from a series of middle and highlatitude rocket flights ( dashed black line ) , and the ca / fe ratio measured by lidar at 54n ( dashed blue line ) , from which the ca / fe ratio is estimated by assuming chemical steady state ( black solid line ) . the ci ratio of the metals ( red dashed line ) and the modeled meteor ablation ratio ( green dashed line ) are shown for comparison . these two profiles should be compared with the chondritic ci ratio ( red dashed line ) [ asplund et al . , 2009 ] , and the ratio of the meteoric ablation fluxes of ca and fe , calculated by applying the chemical ablation model to an astronomical model of cosmic dust [ carrillosanchez et al . , 2015 ] . note that the ablation ratio is a factor of 7 times smaller than the ci ratio because of the differential ablation of the two elements . the [ ca]/[fe ] ratio falls between the ci and the ablation ratios , and the [ ca]/[fe ] ratio is significantly below even the ablation ratio . since the rates of reactions r1r4 in the mlt are reasonably fast between 90 and 100 km , and the dominant forms of these metals in this region of the atmosphere are neutral and ionized atoms [ plane et al . , 2015 ] , the kinetic steady state approximation can be applied to ca and cao : ( e6)dca+dt = k1no+cak2o3ca++k4ocao+0 ( e7)dcao+dt = k2o3ca+k3ecao+k4ocao+0 hence , ( e8)ca+ca = k1no+k3e+k4ok2k3o3e since k 4[o ] is generally more than 3 orders of magnitude larger than k 3 [ e ] between 90 and 100 km [ plane et al . , 2015 ] , ( e9)ca+cak1no+k4ok2k3o3e dividing equation ( e9 ) by the analogous equation for [ fe]/[fe ] , the atmospheric constituents [ no ] , [ o3 ] , [ o ] , and [ e ] cancel . rearranging , and using the rate coefficients listed in table 2 , [ ca]/[fe ] = 8.3[ca]/[fe ] . when the lidar measurement of the [ ca]/[fe ] ratio is multiplied by this factor , the estimated [ ca]/[fe ] ratio ( solid black line in figure 2 ) is seen to be in good agreement with the ion ratio measured by mass spectrometry . inspection of table 2 shows that there are two reasons for this large increase in the ratio of the ions compared with the neutrals . first , charge transfer between no and ca is ~5 times faster than for fe . rate coefficients required to estimate [ ca]/[fe ] from [ ca]/[fe ] we now examine whether the mechanism for sporadic metal layer formation in a descending sporadic e layer [ cox and plane , 1998 ] can explain the appearance of cas below 100 km . figures 3a and 3b show the lidar observations of ca and ca on 9 august 1995 at juliusruh on the island of rgen , germany ( 55n ) . in figure 3a , a pronounced layer of ca ions , confirmed by ionosonde measurements to be part of a sporadic e layer ( shown with white crosses in the figure ) , descends from 100 km to 93 km at a rate of ~4 km h. figure 3b illustrates the simultaneous neutral ca measurements , with the background ca layer continuously present with a peak around 87 km . note that a cas layer appears at 0140 lt once the ca layer has descended to 94 km . in contrast , the patches of relatively high concentrations of ca above 95 km that occur around 0100 and 0200 lt are not accompanied by significant concentrations of ca . lidar observations of ( a ) ca and ( b ) ca on 9 august 1995 at juliusruh , germany . the white crosses in figure ( c ) modeled production of ca from the ca ions in a sporadic e layer whose descent is indicated by the dashed white line . we now use the model of cox and plane to simulate the production of ca in this descending sporadic e layer . the sporadic e layer is initialized with the measured ca density of 810 cm and fe , mg , na , and si densities which are scaled to this using their relative abundance factors measured by kopp . the total metal ion ( and hence electron ) density at 105 km is then 8.9 10 cm , consistent with the ionosonde electron density measurements which ranged from ( 4.5 to 10.5 ) 10 cm between 2300 and 0000 lt . the layer is set to have a full width at half maximum of 1.5 km . note that the sporadic layer is assumed to have a large horizontal homogeneous extent so that advection does not play a role while the layer is descending . the ionmolecule chemistry of ca from the present study and the other four meteoric metals [ plane et al . , 2015 ] are included in the model , along with background atmospheric densities of o3 , o , total density , and temperature from the 1d general mesospheric model mesomod [ murray and plane , 2005 ] . figure 3c shows the modeled production of ca in the descending sporadic e layer ( the background ca layer is not modeled in this simulation ) . the layer , whose position is indicated by the dashed white line , descends at a constant rate to 93 km where the ions are then dumped , as indicated by the ca observations in figure 3a . the model successfully reproduces the height and absolute concentration of the resulting cas , which is an important test of the reaction kinetics , since inspection of figure 3 shows that only ~10% of the available ca is converted to ca during the observational period . moreover , very little neutral ca is produced until the ion layer descends below 95 km , consistent with figure 3b , because the rate of neutralization of ca depends on [ o3 ] and inversely on [ o ] ( equation e9 ) . since [ o3 ] increases ~100fold between 105 and 93 km , and [ o ] is approximately constant , these ion neutralization rates increase by roughly 2 orders of magnitude . this causes the production of ca to occur at the dump altitude , but not higher up so long as the rate of descent of the sporadic e layer is sufficiently fast . in order to study the ionmolecule chemistry of calcium in the mlt , we assign a t temperature dependence to k 3 , which is typical for dr reactions [ florescumitchell and mitchell , 2006 ] . reaction r3 is 45% slower than the dr reaction of feo at room temperature [ bones et al . , we now examine the consequences of this for explaining the very different ca / fe and ca / fe ratios in the 90100 km region of the atmosphere . the dashed black line shows the [ ca]/[fe ] ratio profile measured by rocketborne mass spectrometry . the mean and standard deviation were calculated from seven flights covering a range of latitudes and local times ( note that the geometric , rather than arithmetic , mean is used because of the small data set ; the geometric mean is defined as x=x1x2xnn ) . the dashed blue line shows the mean [ ca]/[fe ] ratio computed from simultaneous and common volume lidar measurements of ca and fe at khlungsborn , germany ( 54n ) . data from 22 nights ( each with 29 h of measurements ) were used ; the total data set is ~120 h of measurements . the measurements were performed between april 1997 and march 1999 . vertical profiles ( 90100 km ) of the measured ca / fe ratio from a series of middle and highlatitude rocket flights ( dashed black line ) , and the ca / fe ratio measured by lidar at 54n ( dashed blue line ) , from which the ca / fe ratio is estimated by assuming chemical steady state ( black solid line ) . the ci ratio of the metals ( red dashed line ) and the modeled meteor ablation ratio ( green dashed line ) are shown for comparison . these two profiles should be compared with the chondritic ci ratio ( red dashed line ) [ asplund et al . , 2009 ] , and the ratio of the meteoric ablation fluxes of ca and fe , calculated by applying the chemical ablation model to an astronomical model of cosmic dust [ carrillosanchez et al . , 2015 ] . note that the ablation ratio is a factor of 7 times smaller than the ci ratio because of the differential ablation of the two elements . the [ ca]/[fe ] ratio falls between the ci and the ablation ratios , and the [ ca]/[fe ] ratio is significantly below even the ablation ratio . since the rates of reactions r1r4 in the mlt are reasonably fast between 90 and 100 km , and the dominant forms of these metals in this region of the atmosphere are neutral and ionized atoms [ plane et al . , 2015 ] , the kinetic steady state approximation can be applied to ca and cao : ( e6)dca+dt = k1no+cak2o3ca++k4ocao+0 ( e7)dcao+dt = k2o3ca+k3ecao+k4ocao+0 hence , ( e8)ca+ca = k1no+k3e+k4ok2k3o3e since k 4[o ] is generally more than 3 orders of magnitude larger than k 3 [ e ] between 90 and 100 km [ plane et al . , 2015 ] , ( e9)ca+cak1no+k4ok2k3o3e dividing equation ( e9 ) by the analogous equation for [ fe]/[fe ] , the atmospheric constituents [ no ] , [ o3 ] , [ o ] , and [ e ] cancel . rearranging , and using the rate coefficients listed in table 2 , [ ca]/[fe ] = 8.3[ca]/[fe ] . when the lidar measurement of the [ ca]/[fe ] ratio is multiplied by this factor , the estimated [ ca]/[fe ] ratio ( solid black line in figure 2 ) is seen to be in good agreement with the ion ratio measured by mass spectrometry . inspection of table 2 shows that there are two reasons for this large increase in the ratio of the ions compared with the neutrals . first , charge transfer between no and ca is ~5 times faster than for fe . we now examine whether the mechanism for sporadic metal layer formation in a descending sporadic e layer [ cox and plane , 1998 ] can explain the appearance of cas below 100 km . figures 3a and 3b show the lidar observations of ca and ca on 9 august 1995 at juliusruh on the island of rgen , germany ( 55n ) . in figure 3a , a pronounced layer of ca ions , confirmed by ionosonde measurements to be part of a sporadic e layer ( shown with white crosses in the figure ) , descends from 100 km to 93 km at a rate of ~4 km h. figure 3b illustrates the simultaneous neutral ca measurements , with the background ca layer continuously present with a peak around 87 km . note that a cas layer appears at 0140 lt once the ca layer has descended to 94 km . in contrast , the patches of relatively high concentrations of ca above 95 km that occur around 0100 and 0200 lt are not accompanied by significant concentrations of ca . lidar observations of ( a ) ca and ( b ) ca on 9 august 1995 at juliusruh , germany . the white crosses in figure ( c ) modeled production of ca from the ca ions in a sporadic e layer whose descent is indicated by the dashed white line . we now use the model of cox and plane to simulate the production of ca in this descending sporadic e layer . the sporadic e layer is initialized with the measured ca density of 810 cm and fe , mg , na , and si densities which are scaled to this using their relative abundance factors measured by kopp . the total metal ion ( and hence electron ) density at 105 km is then 8.9 10 cm , consistent with the ionosonde electron density measurements which ranged from ( 4.5 to 10.5 ) 10 cm between 2300 and 0000 lt . the layer is set to have a full width at half maximum of 1.5 km . note that the sporadic layer is assumed to have a large horizontal homogeneous extent so that advection does not play a role while the layer is descending . the ionmolecule chemistry of ca from the present study and the other four meteoric metals [ plane et al . , 2015 ] are included in the model , along with background atmospheric densities of o3 , o , total density , and temperature from the 1d general mesospheric model mesomod [ murray and plane , 2005 ] . figure 3c shows the modeled production of ca in the descending sporadic e layer ( the background ca layer is not modeled in this simulation ) . the layer , whose position is indicated by the dashed white line , descends at a constant rate to 93 km where the ions are then dumped , as indicated by the ca observations in figure 3a . the model successfully reproduces the height and absolute concentration of the resulting cas , which is an important test of the reaction kinetics , since inspection of figure 3 shows that only ~10% of the available ca is converted to ca during the observational period . moreover , very little neutral ca is produced until the ion layer descends below 95 km , consistent with figure 3b , because the rate of neutralization of ca depends on [ o3 ] and inversely on [ o ] ( equation e9 ) . since [ o3 ] increases ~100fold between 105 and 93 km , and [ o ] is approximately constant , these ion neutralization rates increase by roughly 2 orders of magnitude . this causes the production of ca to occur at the dump altitude , but not higher up so long as the rate of descent of the sporadic e layer is sufficiently fast . the rate coefficient for the dissociative recombination of cao has been measured for the first time and is found to be ( 3.0 1.0 ) 10 cm molecule s at 295 k. this result has two atmospheric implications . first , the surprising observation that the ca / fe ratio is ~8 times larger than ca / fe between 90 and 100 km can now be explained by the known ionmolecule chemistry of these two metals . second , the rate of neutralization of ca ions in a descending sporadic e layer is fast enough to explain the often explosive growth of sporadic ca layers .
abstractthe dissociative recombination of cao+ ions with electrons has been studied in a flowing afterglow reactor . cao+ was generated by the pulsed laser ablation of a ca target , followed by entrainment in an ar+ ion / electron plasma . a kinetic model describing the gasphase chemistry and diffusion to the reactor walls was fitted to the experimental data , yielding a rate coefficient of ( 3.0 1.0 ) 107 cm3 molecule1 s1 at 295 k. this result has two atmospheric implications . first , the surprising observation that the ca+/fe+ ratio is ~8 times larger than ca / fe between 90 and 100 km in the atmosphere can now be explained quantitatively by the known ionmolecule chemistry of these two metals . second , the rate of neutralization of ca+ ions in a descending sporadic e layer is fast enough to explain the often explosive growth of sporadic neutral ca layers .
You are an expert at summarizing long articles. Proceed to summarize the following text: a surprising finding in human transcriptome analysis is that protein - coding sequences only account for a small portion of the genome transcripts ( 1 ) . the majority of the human genome transcripts are non - coding rnas , in particular , long - non - coding rnas ( lncrnas ) ( 2 ) . normally , lncrnas tend to be less conserved across species and often show low expression levels and high tissue specificity ( 35 ) . thus , at the time they were first found , lncrnas were often considered to be transcriptional noise ( 5 ) . in recent years , accumulating studies have revealed that a number of lncrnas are not transcriptional noise but have important functions , for example , affecting gene transcription , targeting rna polymerase ii , regulating splicing and taking part in epigenetics ( 6 ) . moreover , according to the theory of competing endogenous rna ( 7 ) , lncrnas may functionally interact with a broad range of rna molecules through competitively binding with microrna ( mirnas ) , suggesting that lncrnas may have critical roles in a wide range of biological processes . previous studies produced a large amount of lncrna - related data , including sequences , expression profiles and functions . the lncrnadb database provides detailed lncrna information , including sequences , functions , expressions , associated proteins and cellular locations ( 8) . although the noncode database is not specific to lncrna , it curates the sequences , functions , expressions and cellular location of lncrnas in the third version ( noncode v3.0 ) ( 9 ) . studies have reported that lncrna dysfunctions are associated with a broad range of diseases ( 5 ) , including cancers ( 11 ) , cardiovascular diseases ( 12 ) and neurodegeneration diseases ( 13 ) . for example , lncrna pca3 is a highly prostate cancer - specific molecules and a pca3 score has the potential to be a biomarker for prostate cancer aggressiveness ( 14 ) . the up - regulation of the lncrna hotair is an independent prognostic factor of tumor recurrence in hepatocellular carcinoma patients after liver transplantation ( 15 ) . a study confirmed the high specificity and sensitivity of lncrna uca1 from urinary sediments in the diagnosis of bladder cancer , suggesting that uca1 is a potential biomarker for bladder cancer diagnosis ( 16 ) . ( 17 ) revealed that the lncrna bcar4 can be a potential target for antiestrogen - resistant breast cancer treatment because its forced expression in breast cancer cells leads to cell proliferation in the presence of various antiestrogens and in the absence of estrogen . the above studies indicate that lncrnas may help to understand diseases and help to find potential molecules in disease diagnosis , treatment and prognosis . therefore , the study of lncrna disease associations is becoming one of the most important topics of lncrnas and diseases . disease association database will be helpful in studying the roles of lncrnas in diseases but is still not available . to build such a database , we manually curated lncrna disease relations experimentally reported in the literature and created a database , lncrnadisease . moreover , we curated and annotated experimentally supported lncrna interacting partners . in addition , we developed a bioinformatic method to predict novel lncrna first , we downloaded pubmed data , information on non - protein - coding rna genes , and data on gene we further normalized the names of lncrnas and diseases . in total , we curated 166 diseases , of which cancer ( 39.8% ) , cardiovascular disease ( 10.8% ) and neurodegeneration disease ( 8.4% ) were the top three classes ( figure 1a ) . moreover , we provided detailed descriptions for the associations of lncrnas and diseases and curated the dysfunction type for each entry . for example , if an entry s dysfunction evidence is derived from expression data , the dysfunction type of this entry will be considered as expression. the distribution of the dysfunction type is shown in figure 1b . aside from lncrna disease association data , we also curated experimentally supported lncrna interactions and cataloged the interactions according to the interacting molecules and the characteristics of the interactions . for example , at the rna level , lncrnas may interact with proteins ( 18 ) , rnas ( 19 ) , lncrnas ( 20 ) and mirnas ( 21 ) . their interactions may be binding , regulation and co - expression . at the dna level , promoters of lncrna genes may bind with transcription factors ( tfs ) and be regulated by tfs ( 22 ) . figure 1.statistics and distributions of diseases ( a ) and dysfunction types ( b ) of lncrnas in the lncrnadisease database . statistics and distributions of diseases ( a ) and dysfunction types ( b ) of lncrnas in the lncrnadisease database . all data were organized in the lncrnadisease database using sqlite , a lightweight database management system . lncrnadisease was designed not only as a resource for experimentally supported lncrna disease association data , but also as a platform for predicting novel lncrna disease associations . in this study , we present a method to predict novel lncrna we previously showed that mirnas located closely to each other in the genome ( particularly mirnas within 2 kb ) and tend to be associated with similar diseases ( 23,24 ) . here , we investigated whether or not lncrnas tend to be associated with a similar disease as their genomic neighbor genes . thus , we identified the protein - coding genes and mirnas within 2 kb nts of any lncrna with reported disease associations . we then identified the lncrnas with the same associated disease as the neighbor genes / mirnas . we found 33 lncrnas associated with the same disease as their neighbor genes / mirnas . to evaluate the significance , we randomly re - permuted the disease associated with the lncrnas for 10 000 times and counted the number of lncrnas associated with the same associated disease as their neighbor genes / mirnas . as a result , none of the counts was greater than 33 and the expected number was 9 , indicating that lncrnas and their neighbor genes / mirnas tend to be associated with the same disease ( p < 1 10 , randomization test ; figure 2 ) . this result suggests that we can predict potential - associated disease for lncrna through the disease associated with its neighbor genes / mirnas . disease associations and identified potential - associated diseases for all lncrnas identified in the human genome using this tool . finally , we integrated the tool and the predicted results into the lncrnadisease database . blue triangles indicate the distributions of numbers of lncrnas associated with the same disease as their neighbor genes / mirnas in random cases . the red arrow indicates the real number of lncrnas associated with the same disease as their neighbor genes / mirnas . blue triangles indicate the distributions of numbers of lncrnas associated with the same disease as their neighbor genes / mirnas in random cases . the red arrow indicates the real number of lncrnas associated with the same disease as their neighbor genes / mirnas . first , users can browse the lncrnadisease by lncrna names or disease names . when clicking one lncrna or disease in the second , we provide a fuzzy search function for the entries by the full or partial names of lncrnas or diseases in the search page . moreover , all data in the database , including lncrna disease associations , predicted lncrna disease associations and lncrna interactions , can be downloaded . in addition , a detailed tutorial for the usage of the database is available in the the lncrnadisease database will update the experimentally supported lncrna disease association data every 2 months . meanwhile , some new tools for analyzing lncrna disease association data is being developed and will be integrated into the lncrnadisease database in the future . for example , we are developing expression profile- and interacting partner - based methods to predict novel lncrna disease associations and expect to integrate these methods into the database in the near future . increasing studies have shown that lncrnas have important functions and are associated with a broad range of diseases . lncrnas are becoming novel potential molecules for disease diagnosis , treatment and prognosis . in this article the lncrnadisease database integrated several types of data , such as experimentally supported lncrna disease association data , experimentally supported lncrna interaction data and predicted lncrna disease association data . moreover , we developed a bioinformatic method to predict potential - associated disease for a novel lncrna based on its genomic context and integrated this method into lncrnadisease . disease associations are expected to be published in the future and these data will be integrated into the lncrnadisease database . more importantly , although thousands of lncrna have been identified , only a limited number of lncrnas have been reported to be associated with diseases . it is increasingly needed to predict potential - associated diseases for lncrnas through bioinformatic methods . therefore , another major aim of lncrnadisease is to develop and integrate more bioinformatic methods for analyzing and predicting lncrna disease associations . finally , we believe that lncrnadisease is useful for the studies of lncrnas and diseases , and will provide more helps in this topic when it integrates more data and tools in the future . national basic research program of china [ 2012cb517500 ] ; national natural science foundation of china [ 31000585 and 11021161 ] . funding for open access charge : national basic research program of china [ 2012cb517500 ] .
in this article , we describe a long - non - coding rna ( lncrna ) and disease association database ( lncrnadisease ) , which is publicly accessible at http://cmbi.bjmu.edu.cn/lncrnadisease . in recent years , a large number of lncrnas have been identified and increasing evidence shows that lncrnas play critical roles in various biological processes . therefore , the dysfunctions of lncrnas are associated with a wide range of diseases . it thus becomes important to understand lncrnas roles in diseases and to identify candidate lncrnas for disease diagnosis , treatment and prognosis . for this purpose , a high - quality lncrna disease association database would be extremely beneficial . here , we describe the lncrnadisease database that collected and curated approximately 480 entries of experimentally supported lncrna disease associations , including 166 diseases . lncrnadisease also curated 478 entries of lncrna interacting partners at various molecular levels , including protein , rna , mirna and dna . moreover , we annotated lncrna disease associations with genomic information , sequences , references and species . we normalized the disease name and the type of lncrna dysfunction and provided a detailed description for each entry . finally , we developed a bioinformatic method to predict novel lncrna disease associations and integrated the method and the predicted associated diseases of 1564 human lncrnas into the database .
You are an expert at summarizing long articles. Proceed to summarize the following text: anemia is a major health problem globally , which can cause a vicious impediment on quality of life , mortality , morbidity , and socioeconomic progress of a country . it affects more the developing than the developed countries.1 anemia is the most common hematologic abnormality among hiv - infected individuals ; this includes those who are taking antiretroviral treatment ( art ) and those who are art - nave.2 anemia among hiv patients can lead to impaired physical functioning , psychological distress , poor quality of life , accelerated disease progression , and shorter life expectancy.3 although anemia can occur at any stage of hiv infection , its severity is correlated with progression of the hiv disease stage.4 a study conducted in the very early years of hiv discovery had shown the overall prevalence of anemia to be about 28% among people with hiv infection in the pre - aids stage of the disease , whereas it could reach as high as 71% in the advanced or aids stage of the hiv disease.5 according to reports , the prevalence of anemia among adult hiv patients taking art ranges between 23% and 50% globally and 24% and 58% in africa.69 generally , the prevalence of anemia tends to be higher in art - nave patients compared to art users . in ethiopia , the prevalence of anemia among art - nave adult hiv patients was reported to reach up to 35%.10 a study done 3 years ago in the southwest part of ethiopia has shown the prevalence of anemia to be 29.9% and 16.2% among art - nave and art - experienced hiv patients , respectively.11 another study conducted 3 years ago in southern ethiopia has shown the prevalence of anemia among art - nave adult hiv patients to be 23.4% , and the prevalence after 6-month course of art was only 12%.12 various reports have identified different factors to have association with anemia among adult hiv patients including gender , residence , marital status , educational status , income , duration of art , type of art regimen , history of anti - tuberculosis ( tb ) drug treatment , presence of opportunistic infections ( ois ) , advanced stage of the hiv disease , cd4+t - lymphocyte count < 200 cells/l , white blood cell ( wbc ) count < 4,000 cells/l , and platelets count < 200,000 cells/l.7,1119 however , the reported magnitudes and associated factors are different from one geographic area to another and have shown different time trends as the interplay between the various socioeconomic , health , and nutritional conditions alter in sub - saharan country setting . therefore , this study was aimed at assessing the prevalence of anemia and its associated factors among adult hiv patients who were getting follow - up hiv care at debre - tabor hospital , northwest ethiopia . hospital - based quantitative cross - sectional study was conducted from april 1 to may 30 , 2015 , at debre - tabor hospital located in northwest ethiopia . the hospital is found in amhara regional state in the town called debre - tabor , which is 665 km northwest of the capital city addis ababa . the hospital has a catchment population of nearly 2.3 million people and is one of the hiv - care providing centers in the region . it has an average daily patient flow of 2030 adult hiv patients in the working hours of the hiv - care clinic of the hospital excluding emergency presentations on duty hours . the sample size was calculated based on single population proportion formula using a confidence interval ( ci ) of 95% and previous prevalence of anemia being 35% among art - nave hiv patients,10 n=(za2)2p(1p)d2 where , n = sample size , p = prevalence of anemia among art - nave hiv patients and d = assumed marginal error . finally , a minimum sample size of 385 was calculated after anticipating a 10% non - response rate . simple random sampling technique was used to recruit 385 patients charts from a total of 689 . the study populations were all adult hiv patients above the age of 18 years who were on hiv follow - up care at debre - tabor hospital during january 1 , 2010 to december 30 , 2014 , regardless of their art status . those adult patients with incomplete information for hemoglobin ( hb ) status on the chart , either at baseline or during follow - up ; pregnant women ; and women in the post - partum period the questionnaires were constructed with sociodemographic and economic characteristics ; hematological characteristics ; and disease stage , medication treatment , and co - morbidity status - related characteristics . a total of four health professionals , three art nurses as data collectors , and one public health officer as supervisor were recruited for the data collection process of this study . the 2011 world health organization ( who ) report on hb concentration level to diagnose anemia was used in the hospital.20 accordingly , anemia for males was defined as hb concentration < 13 g / dl ( 11.012.9 g / dl = mild ; 8.010.9 g / dl moderate , and < 8.0 g / dl = severe ) , whereas anemia for females was defined as hb < 12.0 ( 11.011.9 g / dl = mild , 8.010.9 g / dl = moderate , and < 8.0 g / dl = severe ) . the data collection tool was pre - tested without hb status determination on 5% of sample size charts of hiv / aids patients before the actual data collection period . all questionnaires were checked during the data collection period by the responsible supervisor on daily basis to check completeness , clarity , and consistency . before analysis , the data were cleaned thoroughly to check for errors during entry . data were entered into epidemiological information ( epi info ) , version 7.1 and analyzed using the statistical package for social sciences ( spss ) version 20 . descriptive statistics , including frequencies and proportions , was used to summarize the study variables . univariable and multivariable logistic regression was used for factors associated with hb status of the study subjects . those variables with a p - value of < 0.2 in the univariable analysis were exported to multivariable analysis to control the possible effect of confounders . the adjusted odds ratio ( aor ) at a 95% confidence interval and p - value of 0.05 was used to declare the statistical significance in the multivariable analysis . ethical clearance was obtained from the institutional review board of the institute of public health of university of gondar . permission letter for the next steps was secured from the debre - tabor hospital administrative body . written informed consent was obtained from participants , and the confidentiality of information obtained was maintained by coding and restricting access to the questionnaire . hospital - based quantitative cross - sectional study was conducted from april 1 to may 30 , 2015 , at debre - tabor hospital located in northwest ethiopia . the hospital is found in amhara regional state in the town called debre - tabor , which is 665 km northwest of the capital city addis ababa . the hospital has a catchment population of nearly 2.3 million people and is one of the hiv - care providing centers in the region . it has an average daily patient flow of 2030 adult hiv patients in the working hours of the hiv - care clinic of the hospital excluding emergency presentations on duty hours . the sample size was calculated based on single population proportion formula using a confidence interval ( ci ) of 95% and previous prevalence of anemia being 35% among art - nave hiv patients,10 n=(za2)2p(1p)d2 where , n = sample size , p = prevalence of anemia among art - nave hiv patients and d = assumed marginal error . finally , a minimum sample size of 385 was calculated after anticipating a 10% non - response rate . simple random sampling technique was used to recruit 385 patients charts from a total of 689 . the study populations were all adult hiv patients above the age of 18 years who were on hiv follow - up care at debre - tabor hospital during january 1 , 2010 to december 30 , 2014 , regardless of their art status . those adult patients with incomplete information for hemoglobin ( hb ) status on the chart , either at baseline or during follow - up ; pregnant women ; and women in the post - partum period the questionnaires were constructed with sociodemographic and economic characteristics ; hematological characteristics ; and disease stage , medication treatment , and co - morbidity status - related characteristics . a total of four health professionals , three art nurses as data collectors , and one public health officer as supervisor were recruited for the data collection process of this study . the 2011 world health organization ( who ) report on hb concentration level to diagnose anemia was used in the hospital.20 accordingly , anemia for males was defined as hb concentration < 13 g / dl ( 11.012.9 g / dl = mild ; 8.010.9 g / dl moderate , and < 8.0 g / dl = severe ) , whereas anemia for females was defined as hb < 12.0 ( 11.011.9 g / dl = mild , 8.010.9 g / dl = moderate , and < 8.0 g / dl = severe ) . the data collection tool was pre - tested without hb status determination on 5% of sample size charts of hiv / aids patients before the actual data collection period . all questionnaires were checked during the data collection period by the responsible supervisor on daily basis to check completeness , clarity , and consistency . before analysis , the data were cleaned thoroughly to check for errors during entry . data were entered into epidemiological information ( epi info ) , version 7.1 and analyzed using the statistical package for social sciences ( spss ) version 20 . descriptive statistics , including frequencies and proportions , was used to summarize the study variables . univariable and multivariable logistic regression was used for factors associated with hb status of the study subjects . those variables with a p - value of < 0.2 in the univariable analysis were exported to multivariable analysis to control the possible effect of confounders . the adjusted odds ratio ( aor ) at a 95% confidence interval and p - value of 0.05 was used to declare the statistical significance in the multivariable analysis . ethical clearance was obtained from the institutional review board of the institute of public health of university of gondar . permission letter for the next steps was secured from the debre - tabor hospital administrative body . written informed consent was obtained from participants , and the confidentiality of information obtained was maintained by coding and restricting access to the questionnaire . we did not include the planned sample of 385 patients charts due to incomplete documentations identified in some charts . therefore , a total of 377 patients chart were reviewed in this study out of which 237 ( 62.9% ) were from patients taking art and 140 ( 37.1% ) were art - nave . more than 60% of the study participants were female , and the mean age of the study subjects was 35.21 with a standard deviation ( sd ) of 9.27 years . three hundred and seventy - five ( 99.5% ) of the participants were amhara by ethnicity and 367 ( 97.3% ) were orthodox christian by religion . a total of 291 participants ( 77.2% ) were urban residents and 191 ( 50.5% ) were married ( table 1 ) . eighty - seven participants ( 23% ) were found to have anemia out of whom 61 were among the 112 art - nave patients making the prevalence of anemia in this group 54.5% . one hundred twenty - six ( 33.4% ) participants had a cd4 + t - lymphocyte count < 200 cells/l . seventy - six ( 20.2% ) study participants had a platelet count of < 200,000 cells/l . nearly three fourths of patients taking art were found to have a normal nutritional status ( table 2 ) . hundred and ninety - one ( 50.7% ) of the participants have had ois , and < 7% of the participants were found to be at the fourth stage of who s clinical disease classification . one hundred and twelve ( 29.7% ) of the study participants were taking zidovudine ( zdv ) containing art regimen . more than 90% of the study participants had no history of anti - tb treatment ( table 3 ) . in the bivariate logistic regression analysis , educational status , types of art regimen , history of treatment with anti - tb drugs , duration of art usage , history of oi , who ; zdv , zidovudine . stage of hiv disease , cd4 + t - lymphocyte count , wbc count and platelets count were significantly associated with anemia . in the multivariate binary logistic regression analysis , type of art regimen , art - usage status , history of treatment with anti - tb drugs , and cd4 + t - lymphocyte count remained to be significantly associated with anemia ( table 3 ) . being art - nave was found to be associated with anemia ( aor : 3.37 ; 95% ci : 1.59 , 7.14 ) . having treatment history with anti - tb drug was also found to be associated with anemia ( aor : 3.2 ; 95% ci : 1.19 , 8.67 ) . taking zdv - containing regimen was found to be associated with anemia ( aor : 2.1 ; 95% ci : 1.03 , 4.57 ) . having a recent cd4 + t - lymphocyte count of < 200 cells/l was also found to be associated with anemia ( aor : 2.1 ; 95% ci : 1.04 , 4.36 ) . our study has demonstrated a high overall prevalence ( 23% ) of anemia among hiv patients who are art - nave and taking art . this finding is concordant with a study done 3 years ago in the southwest ethiopia ( 23.1%);13 similar findings were reported from studies in india ( 23%)6 and ghana ( 24%).8 the prevalence was lower compared to the a study done 3 years ago from northwest ethiopia ( 35% ) ; however , that study included only art - nave hiv patients.10 another study also done 3 years ago in southern ethiopia reported the prevalence of anemia to be 23.4% among art - nave adult hiv patients and only 12% after 6-month course of art.12 studies in indonesia7 ( 49.6% ) , nigeria9 ( 57.5% ) , and uganda18 ( 47.8% ) have reported a much higher prevalence of anemia compared to our study . however , in addition to the sociodemographic differences , those reports reflected the prevalence of anemia among art - nave patients . in our study where two thirds of the participants were taking art , it is expected to get lower prevalence . various studies have pointed out multiple potential factors associated with anemia among hiv patients.7,1119 being art - nave was found to be associated with anemia in our study . this finding is in agreement with the reports from southern ethiopia,12,13 ghana,8 and south africa.11 this can be explained by the various direct and indirect effects of the effective art . one plausible explanation is the fact that art can suppress hiv , a virus which is known to directly affect the bone marrow ; therefore , by suppressing the viral load art could prevent anemia . the other explanation could be related to the indirect effect of art , which is expected to improve the immunity of hiv patients thereby decreasing the occurrence of multiple ois , which are identified to potentially cause anemia . type of art regimen , zdv - containing regimen , was also found to be associated with anemia among hiv patients in this study . this finding is in concordance with reports from addis ababa,15 cambodia,19 and iran.16 zidovudine ( zdv ) has long been identified to be a potential cause of anemia in hiv patients especially early in the art initiation and with low baseline hb.19 additionally , a history of treatment with anti - tb drugs was significantly associated with anemia in our study . a similar finding was reported from the study done in iraq.16 since tb is known to cause anemia through various mechanisms including anemia of chronic illness , bone marrow involvement , malnutrition and hemoptysis , the presence of tb treatment history could partly suggest susceptibility of those hiv patients for anemia from the tb . moreover , the anti - tb drugs such as isoniazid are identified to cause anemia . the cd4 + t - lymphocyte count , < 200 cells/l , was found to be associated with anemia . the possible explanation for this association includes the fact that hiv patients with low cd4 + t - lymphocyte count are known to be at risk of multiple ois , which are known to cause anemia , and the fact that such patients are also likely to have high hiv viral load which could lead to viral infiltration of the bone marrow subsequently causing anemia . since the causes and types of anemia in hiv patients are multi - factorial , we recognize the inability of this study to identify the specific types and specific causes of anemia as a limitation . anemia continues to be a major co - morbidity among adult hiv patients in ethiopia . adult hiv patients who are taking zdv - containing art , with a history of tb treatment , have low cd4+t - lymphocytes count and art - nave should be carefully screened and treated for anemia . we recommend further longitudinal studies to determine predictors of anemia in the setting and intervention programs to change the existing situation .
backgroundanemia is a major public health problem in hiv patients around the world . it has a negative effect on the quality of life of hiv patients and progression of the hiv disease . in the sub - saharan african setting , including ethiopia where both hiv infection and under - nutrition are expected to be high , there is a paucity of data on the matter . this study was aimed to reveal the magnitude and factors associated with anemia among adult hiv patients in debre - tabor hospital , northwest ethiopia.methodsa hospital - based cross - sectional study was used among adult hiv patients in debre - tabor hospital from april 1 to may 30 , 2015 . the diagnosis of anemia was made following the 2011 world health organization recommendation on hemoglobin cut - off points . univariable and multivariable logistic regression was carried out to assess factors associated with anemia.resultsa total of 377 patients charts were reviewed . most of the participants ( n=237 , 62.9% ) were taking antiretroviral treatment ( art ) . the overall prevalence of anemia was 23% ( 95% ci : 19.1 , 27.6 ) . being art - nave ( adjusted odds ratio [ aor ] : 3.37 ; 95% ci : 1.59 , 7.14 ) , having treatment history with anti - tuberculosis ( tb ) drug ( aor : 3.2 ; 95% ci : 1.19 , 8.67 ) , taking zidovudine ( zdv)-containing art regimen ( aor : 2.14 ; 95% ci : 1.03 , 4.57 ) , and having recent cd4 + t - lymphocytes count of < 200 cells/l ( aor : 2.13 ; 95% ci : 1.04 , 4.36 ) were associated with occurrence of anemia among adult hiv patients.conclusion and recommendationanemia continues to be a major co - morbidity among adult hiv patients in ethiopia . adult hiv patients who are taking zdv - containing art , with a history of tb treatment , have a low cd4+t - lymphocytes count and are art - nave should be carefully screened and treated for anemia .
You are an expert at summarizing long articles. Proceed to summarize the following text: its raw or processed roots are the only organ source for clinical uses of traditional chinese medicine , and the leaves are not utilized at all . however , r. glutinosa leaves have the pharmacological effects and can nourish yin , tonify qi and kidney , and promote blood circulation . in the folk , the fresh leaves are often applied externally to treat malignant sore and tinea manus and pedis . as a perennial herb , r. glutinosa has to be annually cultivated and harvested since its fleshy roots easily decay during the winter dormancy or are consumed when new plantlets come out from them in the next season . due to its severe continuous cropping obstacle , however , r. glutinosa plants could not grow well on the same land after the first cropping , decreasing the root yield greatly . thus , exploitation of r. glutinosa leaves not only provides another potential medicinal source for extraction of important bioactive compounds present in its roots but also complements the shortage of r. glutinosa roots due to the limited land and continuous cropping obstacle . a lot of pharmacologically bioactive secondary metabolites [ 4 , 5 ] including iridoid glycosides , phenylethanoid glycosides , and polysaccharides have been isolated in r. glutinosa , of which iridoid glycosides are thought to be main bioactive constituents . till now , more than thirty iridoid glycosides have been separated and identified [ 46 ] , including catalpol , aucubin , geniposidic acid ( gpa ) , rehmanniosides a , b , c , and d , and rehmaglutosides a k . for example , as the main active iridoid glycoside in r. glutinosa , catalpol was found to play the important roles in treatment of many diseases including kidney diseases , neurodegenerative diseases , and diabetes [ 9 , 10 ] . it is revealed that aucubin had pharmacological effects such as antifungals , anti - inflammation and antioxidation , and hepatoprotection . similarly , gpa had therapeutic effects in anti - inflammation , liver disorders , and antinociception [ 14 , 15 ] . among these three compounds , only catalpol was assayed in detail to reveal its spatiotemporal expression profiling among different developmental stages of r. glutinosa leaves and between leaf and root [ 16 , 17 ] . it is shown that catalpol content varied among different developmental stages of r. glutinosa leaves and within the whole growth stage . aucubin and gpa were found to accumulate in roots of r. glutinosa [ 18 , 19 ] , and only the former was quantified and much lower than catalpol in the root [ 18 , 20 ] . therefore , in this paper , we aimed to determine the content of these three iridoid glycosides of two r. glutinosa cultivars in different developmental stages of leaves using hplc - uv method and compared their changing trend . 1 , were grown in around mid - april in 2014 at the implad ( institute of medicinal plant development ) , beijing , china . normal field management was applied during the growth period . on 11th september , 2014 , the top eight positions of leaves from each plant were collected separately , washed to remove the surface soil , and then stored at 80c till use . hplc grade acetonitrile ( acn ) and formic acid were purchased from thermo fisher ( usa ) . the standard catalpol ( 98% ) was obtained from the national institute for food and drug control , china ( http://www.nifdc.org.cn/ ) . standards of aucubin ( gr-133 - 140104 ) and geniposidic acid ( gr-133 - 140423 ) were purchased from nanjing guangrun biotechnology co. ltd . , china ( http://www.grbiology.com/ ) . the concentrations of catalpol , aucubin , and geniposidic acid in the mixed working stock standard solution were , separately , 0.5025 mg , 0.0375 mg , and 0.0020 mg in 1 ml of purified water . to make calibration curves , six series concentrations , ranging from 0.01570~0.5025 mg / ml for ca , 0.001172~0.0375 mg / ml for au , and 6.281e 05~0.002010 mg / ml for gpa , calibration curves were constructed by plotting the logarithm of the hplc - uv peak areas versus the logarithm concentration of each standard . the frozen samples were homogenized with liquid nitrogen and one gram of each sample powder was extracted twice with 25 ml of 30% methanol in an ultrasonic water bath at 25c for 20 min . the supernatants from two cycles of extraction were combined and evaporated to dryness in a rotary evaporator at 50c under the reduced pressure , redissolved in 50 ml of pure water , and then filtered with a 0.22 m millipore membrane filter prior to hplc analysis . for hplc - uv quantification , ten microliters of filtered extracts and standards was run at 30c on a waters 600e system equipped with a phenomenex kinetex c18 column ( 4.6 mm 100 mm , 2.6 m ) , waters 2487 dual wavelength detector , and 2707 autosampler ( usa ) . the isocratic mobile phase contained acetonitrile ( 5% ) and 0.1% formic acid in water ( 95% ) and was delivered at 0.4 ml / min . catalpol and aucubin were monitored at 210 nm and geniposidic acid was monitored at 240 nm . the presence of the three iridoid glycosides in r. glutinosa leaves was determined by comparing both retention time and spectral data with those of their corresponding authentic standards . concentrations of three iridoid glycosides in the samples were determined from the below linear standard calibration curves . are not heat - stable , the batch of leaf samples was used to calculate the dry weight of each stage leaf . hplc - uv method has been successfully applied to quantify catalpol ( ca ) , aucubin ( au ) , and gpa in medicinal plants [ 16 , 17 , 2022 ] . the hplc conditions that researchers used varied in terms of the columns , mobile phase system , and detection wavelengths . therefore , we optimized hplc - uv conditions in terms of column type , uv wavelength , mobile phase composition , addition of formic acid in aqueous phase , and ratio of acn and water in our early work ( table 1 ) . several uv wavelengths ( e.g. , 203 nm , 205 nm , 206 nm , and 210 nm ) were used to detect the presence and content of catalpol and ( or ) aucubin . we found that there was no significant difference in detecting these two iridoid glycosides under 203~210 nm and thus chose 210 nm as the detection wavelength , which was used in china pharmacopoeia ( 2010 ) . there was no much difference of the wavelengths ( 230~240 nm ) in peak shape and separation of gpa except that less miscellaneous peaks and stable baselines were produced at 240 nm . as ji et al . revealed , adding or not adding formic acid ( fa ) in the mobile phase did not obviously influence the peak shape and separation of catalpol and aucubin , except for gpa . while investigating the difference of different concentrations of acn in the mobile phase system on the chromatogram , we found that the elution times of the analytes decreased with acn concentration increased ( table 2 ) . when acn was higher than 5% , catalpol peak was overlapped with the solvent peak . however , when less than 5% of acn was used in the mobile phase , more late au and gpa were eluted . all the three analytes can be eluted within 10 minutes under 5% of acn . in order to shorten the running time for compounds behind them the final hplc conditions that we adopted to determine ca , au , and gpa in our study were summarized in table 1 . using the optimized conditions , we further tested our hplc - uv conditions by checking its linearity , precision , stability , and recovery ( table 3 ) . figure 1 showed that standards of ca , au , and gpa were eluted separately at 3.412 min , 4.857 min , and 9.850 min . their corresponding compounds in the sample extract were detected at 3.436 min , 4.941 min , and 9.813 min , respectively . the linear regression equations obtained were as follows : ca , y = 0.9707x + 6.7723 , r = 0.9997 ; au , y = 0.9952x + 6.8458 , r = 0.9999 ; and gpa , y = 1.0136x + 7.5797 , r = 0.9996 . the precisions were evaluated as the relative standard deviation ( % , rsd ) and , calculated for ca , au , and pga , were 0.14% , 2.65% , and 2.05% , respectively . 25% , 3.08% , and 2.41% , respectively , which indicated that our extraction method was reproducible . to check the stability of extracts , the same sample extract was assayed separately at 0 , 2 , 4 , 8 , 12 , 16 , and 24 h after extraction . the rsd values for ca , au , and pga were 1.46% , 2.38% , and 0.43% , respectively , indicating that our sample extract remained stable at least till 24 hours . the extraction recovery was determined by comparing the content of the compound extracted from the samples with the content of compound from nonextracted standard solutions at equivalent concentrations . to test the sample extraction recovery , one ml of the standard solution containing 4.824 mg of ca , 0.225 mg of au , and 0.0134 mg of gpa , respectively , the recovery rates for standards ca , au , and pga were 102.59% , 98.29% , and 101.96% , respectively . the calculated lod ( limit of detection ) at s / n > 3 was , separately , 1.3333e 04 mg / ml for ca , 1.8170e 04 mg / ml for au , and 4.9926e 05 mg / ml for gpa . the leaf biomass of r. glutinosa cultivars increased greatly from the seedling stage till 120 dap ( days after plantation ) and then deceased after mid - september ( 140 dap ) , partially due to ongoing senescence of bottom leaves ( figure 2 ) . however , the top eight positions of leaves still stayed green and varied in leaf size , which can represent the different developmental stages of leaves ( figure 3 ) . the 8th leaves , counted downwards from the uppermost leaves , looked similarly in their size and appearance status as the lower positions of unsenescent leaves . however , their dry weight ( dry weight per gram fresh weight ) changed dynamically with leaf development ( figure 4 ) . overall , leaf dry weight decreased with the leaf development and increased for l5 and then decreased in the rest of older leaves . l1l8 had about 0.140.18 grams of dry biomass per gram fresh biomass . in this paper , we just focused on the contents of ca , au , and gpa in the top eight positions of leaves and investigated their relationship with the leaf developmental stages . although the contents of ca , au , and gpa were somehow different between wen 85 - 5 and beijing no . 1 , the changing trends of these three iridoid glycosides with the leaf developmental stage were very similar between the two cultivars , indicating no genetic difference in developmental regulation of these three iridoid glycosides ( figures 5 and 6 ) . generally , catalpol , a major bioactive iridoid glycoside of r. glutinosa , was higher in younger leaves and decreased with the leaf development , which was consistent with the result of ji et al . . likewise in r. glutinosa root , aucubin in the leaf was still much lower than catalpol , which was found by pitczak et al . . the changing trend of aucubin among different developmental stages of r. glutinosa leaves was distinct from catalpol ; that is , the older the leaves , the higher the aucubin in them . it was found that aucubin was one of the intermediates for catalpol biosynthesis in scutellaria albida and paulownia tomentosa . the opposite metabolic profiling of aucubin and catalpol in r. glutinosa leaf indicated that catalpol might not be synthesized via aucubin ; however , this needs to be further investigated . gpa was detected to be present in r. glutinosa leaves and the lowest analyte in all the developmental stages of r. glutinosa leaves . like aucubin , gpa was increased with the leaf development and higher in older leaves . in this paper , we determined contents of catalpol , aucubin , and geniposidic acid in different developmental stages of r. glutinosa leaves using the optimized hplc - uv conditions and further compared their changing trend with the leaf development . our results showed that aucubin and gpa in r. glutinosa leaves were much lower than catalpol and had the increasing trend with the leaf development , which was different from catalpol . this work provided an important basis for future exploitation of r. glutinosa leaves such as isolation of interesting metabolites , for example , the high concentration compound present at 4.16 , which was further identified by hplc - lc - ms method . it also provided a model to study the relationship of aucubin and catalpol metabolisms in r. glutinosa .
although r. glutinosa roots are currently the only organ source in clinics , its leaves are a potential supplement for the roots especially in extraction of some important bioactive compounds . our early work found that the contents of catalpol and total iridoid glycosides varied among different developmental stages of r. glutinosa leaves . aucubin and geniposidic acid , the abundant major bioactive compounds in eucommia ulmoides and gardenia jasminoides , respectively , were found present in r. glutinosa roots , however , and have not been analyzed in its leaves . in this paper , we aimed to determine contents of these three iridoid glycosides in different developmental stages of r. glutinosa leaves using the optimized hplc - uv conditions . our results showed that aucubin and gpa in r. glutinosa leaves were much lower than catalpol and showed the increasing trend with the leaf development , which was different from catalpol . this work provided the important information for future exploitation of r. glutinosa leaves as a potential supplement for its roots in extraction of some important bioactive compounds and studying the relationship of aucubin and catalpol metabolism .
You are an expert at summarizing long articles. Proceed to summarize the following text: as a group , older people are steadily increasing in both numbers and proportions in most countries . approximately 600 million people worldwide are now aged 60 or older a number estimated by the world health organisation to double by 2025 . this global trend is also seen in sweden , where the current population of 1.6 million people aged 65 years or above is projected to increase to 2.2 million by 2025 . the majority of older people in sweden live independently , however , increasing numbers of those living at home are frail and dependent on daily help from others . approximately 80% of the adult population in sweden are enrolled in a regular dental check - up system , of which the public dental service manages 42% . treatment is not free of charge , but the national health insurance covers a certain amount of the expenses . there is also a special dental care allowance for older people receiving substantial supportive care for daily living in their homes or at institutions . under this special dental care allowance , the maximum fee for the elderly is sek 1100 per year ( 115 ) . approximately 6% of the inhabitants aged 65 years and older in the study region are eligible for this special dental care allowance . oral health patterns have changed as increasing numbers of older people retain their own teeth , many of which are heavily restored . older people have an increased risk of developing oral health - related problems , due to impaired saliva secretion associated with medication , changes in dietary habits , and difficulties in managing at - home dental care . thus , providing adequate dental care for older people is a considerable challenge for the dental community . caring for and helping older people maintain good oral health will require special competence and occupy more of the dental team 's time . regular dental check - ups , preventive measures , home care , and dental care are important to maintaining good oral health . many dental clinicians report that their older patients lose contact with their dental service , commonly cancelling or failing to appear for scheduled appointments . a study by strmberg et al . concluded that of the several factors associated with the risk of developing caries among homebound elderly people on moderate or substantial supportive care , one of the most important was not having a dentist . lack of contact with dental services has also been observed on the admission of older people to nursing homes . a study from france in 2000 reported that over 70% of newly admitted residents had not seen a dentist for over 5 years . the main aim of this study was to examine to what extent older people enrolled in a regular dental check - up system lose contact with their dental health service . one additional aim was to record dental status and the use of medication in a subsample of the study group to illustrate the oral and physical health of the study group and compare it with the national figures to be able to comment on the representativeness of the sample . data from electronic dental journals were collected from three public dental clinics in the region vstra gtaland , sweden . region vstra gtaland , situated on the swedish west coast and inlands , is the largest county council in sweden , with a population of 1.6 million , and its mix of urban and rural areas , small communities , and major cities mirrors that of sweden as a whole . two of the communities are fairly small , with approximately 10000 inhabitants each , and are situated approximately 200 km from the county 's main city , gothenburg . the population of the suburb with 19% aged over 65 years is somewhat younger than that of the two smaller communities , each with approximately 25% of the population aged over 65 . each community has one or more private dental surgeries and one public dental clinic serving the adult population . patients included in this study were all enrolled in a dental - recall system at the three selected public dental clinics and were invited for a dental check - up according to their estimated need and individual demand . the study group were aged 65 or older and were living independently or with moderate supportive care for daily living in their own homes . the recall system ensures that patients are scheduled and contacted by the clinic by telephone or post for dental check - ups at least every second year . if the patient declines the invitation or fails to attend the scheduled appointment , it is noted in the record . it is also noted when patients move from the area or choose another dental caregiver . in sweden , information about whether patients have moved or died is available from the regional population registry that is cross - linked with electronic dental records and is updated on a regular basis . these records are maintained according to the standards of the national board of health and welfare . to be included in the main study , excluded from the study were those who had died during the study period;wanted emergency care only;obtained or during the study period became eligible for the special dental care allowance subsidized by the county council ( because such dental care includes a free annual in - home examination by a dental hygienist);lived in or moved to a nursing home during the study period because all residents of nursing homes are entitled to the special dental care allowance;had moved from the community and/or left the clinic for another caregiver during the study period . had died during the study period ; wanted emergency care only ; obtained or during the study period became eligible for the special dental care allowance subsidized by the county council ( because such dental care includes a free annual in - home examination by a dental hygienist ) ; lived in or moved to a nursing home during the study period because all residents of nursing homes are entitled to the special dental care allowance ; had moved from the community and/or left the clinic for another caregiver during the study period . individual data from dental records during 20042009 for 4759 patients were examined longitudinally by one person at each clinic ( two dentists and one dental hygienist ) . the selected patients were 65 years old at the beginning of the study in 2009 . after application of exclusion , the dental records of 3648 participants ( 1690 men and 1958 women ) were included in the study ( small community 1 , n = 912 ; small community 2 , n = 754 ; suburb , n = 1982 ) . distribution of participants according to age group and sex in the study sample the data from the dental records noted in the protocol were age , gender , and active ( at least three examinations in the study period ) versus nonactive ( at least three declined visits ) patient status . to gain an overview of the sample and its representation of the population , a subsample of 1102 patients was randomly chosen from the different age groups in the main sample of 1982 patients from the largest clinic . randomization was conducted by selecting every second individual in each age group , other than the oldest group , from a printed list . because there were fewer participants in the oldest age group , all participants the records from the subgroup were analyzed according to the number of teeth , dentures , implants , medication status , and loss of regular attendance . these data were collected from the last registration in the individual dental records in the study period 2004-2009 . in the two small communities the regional ethical review board of gothenburg approved this study ( decision number 402 - 10 ) . data analyses included descriptive statistics and chi - square linear - by - linear test . the statistical analyses were performed using the ibm spss statistics for macintosh , version 21.0 . data analyses included descriptive statistics and chi - square linear - by - linear test . the statistical analyses were performed using the ibm spss statistics for macintosh , version 21.0 . a clear association between age and loss of regular dental attendance was found among patients aged 65 years and older ( = 114.0 , p < 0.001 ) [ figure 1 ] . between 2004 and 2009 , 13% of the participants lost contact with the dental health service ( 10% in those aged 6579 and 21% in those 80 ) . distribution of participants who had lost contact with the dental health service during the study period according to age group ( n = 3648 ) table 2 shows dental status , use of medication , and loss of regular dental attendance in the subsample . seven percent of the participants had implants and 6.4% had a full denture in one or both the jaws . in total , 12% of the subsample had lost contact with dental services during the study period . oral status ( teeth , implants , and full dentures ) , number of medications , and loss of dental attendance in the subsample group from the largest clinic ( n=1102 ) . this study shows that the proportion of older people who lose their regular contact with dental services increases with age . this is in line with both the reported experience of dental staff and data from other studies . it may seem natural that old people lose contact with dental services as they move to special housing or die . in this systematic study , such factors were treated as exclusion criteria and only those people still living in their own homes , with or without help , were included . to our knowledge , this is the first study to systematically investigate dental attendance in a large sample of older people . our finding of a clear relationship between age and an increasing tendency to lose a formerly regular pattern of dental contact is an important observation . however , old age may not be the only reason for the loss of contact with the dental health service . our results probably reflect a relationship between increasing age and a number of associated factors that reduce the ability of old people to maintain contact with the dental health service . it is likely that difficulty in maintaining contact with dental services coincides with various mental and physical health problems such as incipient dementia and multi - disease conditions that often correlate with age and an increasing dependency on others to cope with everyday life in what is sometimes referred to as the frail period . in addition to mental and physical health problems , other factors such as marital status , socioeconomic and sociocultural contexts , oral status , availability of dental service , and the knowledge and attitude of the dental staff may contribute to whether regular dental contact can be maintained . older adults born before 1940 may not value oral health and dental aesthetics as much as younger generations . it is possible that the patterns of dental contact in smaller municipalities are different from those in cities and towns . one factor that may affect the dental contact pattern is accessibility of care . in urban areas , however , in the three municipalities studied , dental service was easily available , and in smaller communities , dental professionals may have better knowledge of their older patients and their needs for service . social acceptance of oral deficiencies might also be higher in small municipalities , and this may then affect dental care habits , but we found no scientific data to support this hypothesis . social relations have , however , been found to be important to oral status or oral health . people who live alone are more likely to have fewer teeth and more anterior tooth spaces . the strength of this study is that earlier in life all the participants had chosen to enrol in a dental care system with regular recall . in sweden , , we can assume that the participants had reasonably regular visits throughout most of their adult lifespan . a limitation of the method of a retrospective study of patient records is that over time many different people may have been involved in writing the records . despite guidelines for maintaining consistent records , however , prior to the start of the study all three investigators were trained to fill in the protocol based on what was found in the records , so we can expect a consistent interpretation of the records across the three clinics . the sample was drawn from smaller communities , all located in the same county in western sweden , but fairly well correlated with the national population distribution . the study group of 65 years and older represented 25% of the population in the two smaller study communities and 19% in the largest . the corresponding figure is 18% for vstra gtaland and 19% for sweden as a whole . the proportion of older people is generally slightly lower in larger towns and cities than in smaller municipalities . to gain information about representativeness , a subsample of the main sample were randomly selected and their dental records examined for number of teeth , dentures , implants , medication status , and loss of regular attendance . these data were collected from the last registration in the individual dental records during the study period 20042009 . according to the national board of health and welfare , people aged 6090 in sweden have an average of 21 remaining teeth . seven percent of our subsample had one implant or more , a proportion quite similar to the national figure of 5% . thus , the patients in our study correlated quite well with the elderly population of sweden or parts of sweden with regard to number of teeth and implants . in a repeated cross - sectional study of the population in a swedish county , the proportion of totally edentulous 80-year - old individuals had decreased from 56% in 1983 to 5% in 2003 and even lower in 2013 . the corresponding figure for sweden is 5 to 6 medicines for the group 75 years and older . a recent study showed that elderly people ( 75 years and older ) dependant on moderate and substantial supportive care used approximately six medicines , but our study group was somewhat younger , and in general probably healthier because substantial supportive care was a criterion for exclusion . the findings of this study showing a clear relationship between age and an increasing tendency to lose a formerly regular pattern of dental contact support the results of telephone interviews collected by the living conditions survey ( scb statistics , sweden ) , which showed that people aged 16 years and older reported declining regular dental contacts with increasing age . the ability of old people to maintain contact with their dental health service seems , therefore , often to be reduced . patient age is therefore an important indicator to consider in the planning of the oral health care system . lack of dental contact is likely to escalate the risk of oral health problems , which , in turn , may have negative consequences for patients ' quality of life during their later years . the frail period of life probably coincides with the time of not seeing the dentist , developing impaired saliva secretion due to medication , changes in diet , and difficulties in oral hygiene , which can result in the rapid deterioration of their dental status and have a significant impact on their oral health - related quality of life . it is very important for older people , dental services , and society as a whole to preserve the good oral health that has been established through a lifetime of regular dental care . the result of the present study shows , however , that a considerable number of older people living independently or with moderate supportive care in their own homes lose contact with dental service despite enrolment in a recall system . in future studies , it will be important to identify the reasons why older adults lose contact with the dental health service . knowing these causes may enable us to develop methods to identify individuals at risk of losing contact with the dental health service and develop procedures to ensure regular dental contacts . the local research and the development board for gothenburg and sdra bohusln supported this study . the local research and the development board for gothenburg and sdra bohusln supported this study .
aim : our objective was to study the loss of dental attendance and a possible age trend among patients aged 65 years in sweden . regular dental check - ups are considered to be an important factor in maintaining oral health . approximately 80% of the adult population in sweden are enrolled in a regular check - up system ; however , dental practitioners often find that older patients attend fewer check - ups . old people may naturally lose contact with dental services as they move to special housing or die . in this systematic study , these factors were investigated and used as exclusion criteria.materials and methods : data were collected for all patients ( n = 4759 ) aged 65 or older from the electronic journal system in 3 large public dental clinics in 3 communities . their dental records for the years 20042009 were studied longitudinally by 1 person at each clinic ; 1111 patients were excluded ( patients died during study period , wanted emergency care only , obtained special dental care allowance , moved from the community or moved to special housing , or left the clinic for another caregiver ) . the statistical analyses were performed using the statistical package for the social sciences version 21 ( ibm).results : of the 3648 patients ( 1690 men and 1958 women ) included in the study , 13% lost contact with their dental service over the course of the study ( 10% of those were aged 6579 and 21% 80 ) . the decrease in regular dental contact had a statistically significant association with increasing age ( p < 0.001).conclusion : a considerable number of older people living independently or with moderate supportive care in their own homes lost contact with dental service despite enrolment in a recall system .
You are an expert at summarizing long articles. Proceed to summarize the following text: population policies in any country are considered as the basis of social - economic development ( 1 ) . these policies encompass all population trends , morbidity and mortality , reproduction and family formation , distribution , migration , and , overall , the size and structure of the population of a country ( 2 ) . of them , fertility is a principal component of population dynamics that total fertility rate ( tfr ) is a common indicator for it ( 3 ) . tfr is the average number of live births a woman would have throughout her life . in the case of tfr<2.1 , the united nations population division has long - term forecasts for it that demonstrates continued fertility declines in the developing world ( 5 ) . shapiro and gebreselassie remark that most countries in sub - saharan africa are still facing fertility declines . they showed in the other regions of the world , every country - with the exception of armenia and indonesia - also is experiencing decreasing fertility . these regions contain both countries that have a modest decline , such as guatemala and india , and those for which the decline is more substantial , for instance morocco , brazil , and yemen ( 6 ) . as a developing country , iran has a tfr = 1.75 births per woman according to the last census of 2011 , which has decreased from the rate of 1.84 in 2006 ( 7 ) . tfr in iran began to decline in 1985 when it was 6.3 births per woman , and , lately , it has decreased markedly ( 8) . despite various fertility levels and patterns in different parts of the country , for example , according to the 2006 census , tfr for urban and rural areas were 1.75 and 2.1 , respectively , as compared to 3 and 4.2 in 1994 . moreover , four provinces had tfr values between 1.2 and 1.6 and 13 had rate between 1.7 and 2.1 ( 9 ) . consequently , the rate of growth of the older population is increasing and is predicted to reach nearly 24% by 2050 ( 10 ) , meaning that the population age structure of the country is converting from youth into aging ( 11 ) . for instance , on economic factors , adsera pointed that unemployment and unstable contracts might reduce fertility significantly by delaying childbearing ( 12 ) . also , it pointed to young couples who significantly postpone their childbearing due to uncertain conditions in the labor market , such as temporary employment contracts or job instability ( 13 ) . caldwell ( 1982 ) asserted that the rising costs of children ( e.g. , for education ) and their declining economic value ( e.g. , for labor and old age security ) result in a decline in the desired family size . this , in turn , leads to an increase in the demand for and the use of birth control ( 14 ) . he showed that rising educational levels make childrearing more expensive than it used to be , which could indicate that wealth flows are changing directions from parents to children ( 15 ) . also , based on a survey by kreyenfeld , economic indicators in the form of unemployment and subjective perceptions of the economic situation ( e.g. , fear of job security ) make highly educated women delay bearing their first child ( 16 ) . in a study by raziyeh shakoori in iran , employment and financial independence of women were the two main causes of divorce and , consequently , the decline in fertility ( 17 ) . social factors are other issues that might affect tfr . for example , in rural communities with the traditional agriculture context , due to high mortality , high fertility prevails to ensure the survival of the population . as a society develops and socio - economic shifts occur , such as increasing education and urbanization , the reduction in child mortality , and the increasing cost of children , the actual fertility decreases ( 18 ) . abbasi - shavazi and asgari ( 2005 ) studied family and fertility changes in iran and indicated that women have fewer children due to the high degree of urbanization and the increasing marriage age and education levels as modern patterns of fertility ( 19 ) . in a study in saudi arabia , it was shown that marriage patterns have been changed by the increasing ages of people when they get married , reducing early marriages in the country , and by improving women s education and decreasing infant mortality , thus , fertility rates have been decreased ( 20 ) . among the indicators of development , there was a significant correlation between degree of urbanization and fertility in bongaarts s work ( 21 ) . hence , understanding the notion of tfr and the factors that affect its decline could present helpful policy insights and implications towards preventing and managing its effects in different societies . despite all of the above - mentioned studies , overall , there are very few studies on the determinants of fertility decline in the developing countries , especially in iran taking a qualitative approach . operationally , we selected four indicators of socioeconomic development , i.e. , divorce , marriage , urbanization , and unemployment rates , to examine their effects on tfr in iran between 2002 and 2012 . this could help to form a detailed and comprehensive understanding of population issues and , thereby , aid in the development of sustainable population policies . the panel data method was used to determine the association between divorce , marriage , urbanization , and unemployment rate and tfr in iran between 2002 and 2012 . required data on variables was obtained from the nocr database and the statistical center of iran . data on tfr for ten years ( 20022012 ) were taken from abbasi - shavazi and hosseini - chavoshi s work ( 22 ) . at this stage , a checklist was developed based on the needed variables to check when we went to the data centers , and , using this tool , we completed it based on different provinces . it should be noted that a letter of introduction from the faculty was prepared for them , and , at the same time , some explanations were provided to them about the topic of the study and its importance to ensure their cooperation . to determine a suitable model for estimating panel data , likelihood ratio and huasman tests were done using eviews 7 software . in the likelihood ratio test , in contrast , a rival hypothesis shows the existence of the fixed effect . in the huasman test , the null hypothesis also displays the non - correlation between the disturbance term and exogenous variables . the rival hypothesis of the huasman test indicates that an appropriate model is the random effect regression ( 23 ) . correlation among the study variables was shown by capturing this function : tfr= f ( mar , div , u , ur ) ; where mar : marriage rate , div : divorce rate , ur : urbanization rate , and u : unemployment rate . the proper regression model was given by : ltfr = 0 + 1ldivit + 2lmarit + 3lurit + 4luit + uit the panel data method was used to determine the association between divorce , marriage , urbanization , and unemployment rate and tfr in iran between 2002 and 2012 . required data on variables was obtained from the nocr database and the statistical center of iran . data on tfr for ten years ( 20022012 ) were taken from abbasi - shavazi and hosseini - chavoshi s work ( 22 ) . at this stage , a checklist was developed based on the needed variables to check when we went to the data centers , and , using this tool , we completed it based on different provinces . it should be noted that a letter of introduction from the faculty was prepared for them , and , at the same time , some explanations were provided to them about the topic of the study and its importance to ensure their cooperation . to determine a suitable model for estimating panel data , likelihood ratio and huasman tests were done using eviews 7 software . in the likelihood ratio test , in contrast , a rival hypothesis shows the existence of the fixed effect . in the huasman test , the null hypothesis also displays the non - correlation between the disturbance term and exogenous variables . the rival hypothesis of the huasman test indicates that an appropriate model is the random effect regression ( 23 ) . correlation among the study variables was shown by capturing this function : tfr= f ( mar , div , u , ur ) ; where mar : marriage rate , div : divorce rate , ur : urbanization rate , and u : unemployment rate . the proper regression model was given by : ltfr = 0 + 1ldivit + 2lmarit + 3lurit + 4luit + uit the urbanization percentage of our country was high , and the mean of tfr was estimated to be lower than the substitution threshold in the country . before econometrics estimation , the stationary level of variables should be determined . in the literature on econometrics , regression of a non - stationary variable upon another non - stationary variable , levin , lin , and chu s unit root test was run to explain stationary variables ( table 2 ) . the results in table 2 reflect that all variables were first - order - integrated . in other words , by once differencing , all variables convert to the stable level . an important exception arises when two or more one - order , integrated variables are co - integrated , i.e. , if there is a particular linear combination of these non - stationary variables that is stationary . the results of the pedroni test showed that the variables were co - integrated and that they had long run linkage ( p < 0.05 ) . different models were introduced to estimate the panel data . based on the econometrics literature , three models were specified , i.e. , proper tests were used to choose the best model . to diagnose between the pooled and fixed effect regression , we used the redundant fixed effects test , which rejected the null hypothesis ( p < 0.05 ) . hence , we could say there is a cross section fixed effect . the next step was to determine the type of effects . for assessing the effect behavior , results of estimating the fixed model are shown in table 3 , which indicates that the divorce rate has an adverse effect on tfr . based on the results , except the divorce rate and marriage rate , effects of other studied factors on tfr were not confirmed . the results of this study compared to those that have been done in this area presented below . over the past 25 years , we have witnessed dramatic increases in the number of divorces in the country , so that the crude divorce rate of 0.7 has reached to 1.9 ( per 1000 population ) over the period of 19862011 ( 24 ) . in our analysis , a significant and adverse relationship was observed between divorce rate and tfr . in other words , a one percent increase in the divorce rate results in a 0.03% decrease in tfr , on average . with the decrease in the durability of marriages all of these could have significant effects on tfr ; so that , this adverse association was confirmed based on the present study . in a study within the country , employment and financial independence of women were considered as causes of divorce and , thus , fertility decline ( 17 ) . lee and bumpass ( 2008 ) concluded that , if a woman s work is irregular and takes long hours of her time , even though her income is very high , it does not help the stability of the family ( 25 ) . another variable that we examined in relation to the decrease in tfr was the marriage rate . it had significant relationship with tfr , so that a one percent increase in marriage rate increased tfr by 0.04 percent , on average . abdul salam , in his study , showed that marriage patterns have changed by reducing early marriage and improving women s education , marriage age , and reducing infant mortality and children cause to reduce the number of childbirths in saudi arabia ( 20 ) . in this study , this relationship has been investigated in several studies . along with the urbanization and industrialization of societies , the cost of raising children has increased and , because of economic considerations , the average number of children born decreases ( 26 ) . abbasi - shavazi and asgari studied the family changes and fertility decline among 1,249 women aged 15 to 49 in yazd province , iran , and showed urbanization and higher education and higher age of marriage cause people to have fewer children ( 19 ) , but our results do not support this finding . bongaarts , using the fixed effects regression for the period of 196065 to 199095 , expressed that among the development indicators , the gdp per capita and participation of workforce in the agriculture sector did not have a significant relationship with tfr and only urbanization had a significant and inverse relationship ( 21 ) , but this was contrary to our analysis . some have posited that fertility reduction in all provinces of iran , across the rural and urban areas or the convergence of the reduction , has occurred ( 22 ) and that a simultaneous and substantial decrease across all geographic regions and in both urban and rural areas had occurred and there is a considerable degree of narrowing of urban - rural and regional ( 27 ) . hence , there was no significant relationship between urbanization rate and tfr that are consistent with our results . in this study , unemployment rate and tfr showed a negative but statistically insignificant association . women s employment contributes to decline in fertility , and women s entering to labor market is affected by fertility . the childbearing age of women has increased because of changes in the socio - cultural conditions , especially employment ( 28 ) . addio and ercole claimed that , statistically , there was a positive significant relationship between enhanced financial security of women through participation in the labor market and a reverse correlation between the women unemployment with fertility ( 12 , 29 , 30 ) . while the relationship between women s employment and fertility rate in 1970s was adverse , but in the 1990s , it became a positive correlation to the extent that countries with high employment levels for women have higher levels of fertility ( 31 ) . these results are in contrast to studies based on economic model that expresses the fertility of women who are working , because of facing with more opportunity costs of children are in lower fertility levels ( 31 , 32 ) . in this way , kogel s results based on econometric panel data techniques of 21 oecd countries for the period 1960 and 2000 , respectively , showed between fertility and female participation in the labor force , a negative correlation was observed ( 33 ) . the reason may be that due to data constraints about women and men unemployment separately , we used total unemployment in the study according to statistical center of iran data . another reason might be the difference of our context comparing those countries , which are mostly developed . considering our context and situation and importance of employment , especially for men , there are some important explanations about the relationship between unemployment and fertility . the first point turns to the importance of job security that is much more essential than job creation . if government generates some jobs , almost temporary or contractual cases , families postpone their childbearings and reduce them , because of the uncertainty of their future career . if household income is low compared with its expenditure , due to lack of sufficient financial ability or wage to cover the costs of childbirth , family could not raise fertility . final note is associated to the provision of facilities for pregnant mothers and then facilities such as kindergartens for children of employees . if the system does not offer such facilities , a person with a job and even job security might still have lower fertility . although , this study in terms of using rich data from several census as well as longitudinal data collected from various bodies might have important and useful points for policy - makers and officials but like any other study has limitations and due to limited available data , a group of variables such as culture and values were excluded . in addition , since the provincial data were collected over a long period , so for a number of provinces in the period in question , the data were not separated , so we combined data from these cases . furthermore , urbanization rate and unemployment rate did not have significant relationships with tfr . due to the less effect of some socio - economic factors on tfr , it could be concluded that other variables outside of the studied factors , such as cultural and value - related issues in addition to social economic factors , might play a role in this area and have more effect and prominent role on tfr decrease . preparing the ground for sustainable employment and improving job security in the future , along with providing the facilities , such as insurance , housing for adults are suitable suggestion . in addition , providing the necessary skills in family for mate selection to have better durability as well as emphasizing communication , emotional , cultural skills after marriage as very important topics could be helpful in this area . finally , emphasis on family roles in marriage procedures that would be much lower divorce . it should be noted that , despite the common perception that the socio - economic factors in islamic republic of iran are major causes of reduced fertility , the results of our study show that other factors , including cultural values and contexual values , might have greater effects . thus it is recommended that more studies be conducted focused on the effects of such factors .
introductionover the last few decades , total fertility rate ( tfr ) has followed a downward trend in iran . the consequences of this trend from the perspectives of some are negative . considering the macro - population policies in recent years , this study aimed to examine the effect of some macro socio - economic variables , including divorce , marriage , urbanization , and unemployment rate on tfr in iran from 2002 to 2012.methodsthis time series research was conducted in 2015 using the databases of the national organization for civil registration ( nocr ) and the statistical center of iran . the study population was the related data of provinces in the selected variables . the main methods used in the research were the common unit root test , pedroni cointegration test , redundant fixed effects tests , correlated random effects - hausman test , and panel least squares of fixed effects . in order to determine the suitable model for estimating panel data , likelihood ratio and huasman tests were done using eviews software , and the fixed effects regression model was chosen as the dominant model.resultsthe results indicated that the divorce rate had a negative and significant effect on tfr ( p < 0.05 ) . a positive and significant relationship between marriage rate and tfr variables also was observed ( p < 0.05 ) . urbanization rate ( p = 0.24 ) and unemployment rate ( p = 0.36 ) had no significant relationship with tfr . according to f statistic , significance of the overall model also was confirmed ( p < 0.001).conclusiondue to the lower effect of the studied factors on the reduction of tfr , it seems that variables other than the ones studied , as well as cultural factors and values , might be fundamental factors for this change in the country .
You are an expert at summarizing long articles. Proceed to summarize the following text: primary care physicians need up to date knowledge on contraceptive counseling for women in order to provide the best match between patient and contraceptive method . additionally , providers frequently need to supply contraceptives to women who have particular medical comorbidities that may be worsened by pregnancy or necessitate the use of potentially teratogenic medications . newer oral contraceptive preparations are now available which differ from traditional oral contraceptives in their hormonal dosages , cycle length , and hormone - free intervals . effective contraceptive counseling requires an understanding of a woman s preferences and medical history as well as the risks , benefits , side effects , and contraindications of each contraceptive method . the newer formulations and extended cycling regimens are attractive options to women for both their contraceptive and non - contraceptive benefits . in this review , we will focus specifically on providing an update on the extended - cycle levonorgestrel - ethinyl estradiol contraceptives . oral contraceptive pills ( ocps ) contain both an estrogen ( ethinyl estradiol , mestranol , or estradiol valerate ) and a progestin . individual oral contraceptive packets differ in terms of their estrogen dosage , progestin type and dosage , cycle length , and hormone free interval . all combined - hormonal ocps in the united states contain ethinyl estradiol ( ee ) , usually at doses between 2030 mcg , but many different progestins are available . levonorgestrel ( lng ) is a widely utilized contraceptive progestin which is found in many combined oral contraceptives but is also highly effective alone in progestin - only pills , long - acting subdermal implants , intrauterine devices , and in emergency contraception . the mechanism of action of lng differs depending on its dose and delivery method ( oral , intrauterine , transdermal ) . the oral bioavailability of lng is approximately 90%100% because it is not subject to first - pass metabolism.1 when lng ( or another progestin ) is used in combination with ee , the primary mechanism of action is prevention of the surge of luteinizing hormone thereby preventing ovulation ; but progestins also thicken the cervical mucus and alter the endometrial lining to help prevent fertilization or implantation . traditional oral contraceptive pill regimens consist of a 21-day course of hormones followed by a 7-day hormone - free interval . this regimen was originally devised to mimic the natural menstrual cycle and help women feel comfortable in accepting the ocp . during the standard 7-day hormone - free interval that occurs with use of low - dose estrogen ocps , the function of the hypothalamic - pituitary - ovarian axis recovers rapidly . this recovery can increase the risk of ovarian follicle development , unintended ovulation , and increased spotting due to endogenous estradiol production.26 fluctuating hormone levels also allow endometrial buildup and can exacerbate endometriosis and premenstrual symptoms ( eg , headaches , tiredness , bloating , excessive bleeding , and menstrual pain ) by creating hormone excess and withdrawal states.36 newer extended - cycle regimens shorten or eliminate the hormone - free interval , decrease frequency of menses to four times per year or eliminate menses altogether . this can reduce the risk of common menstrual symptoms , endometriosis , or severe dysmenorrhea by offering potentiallygreater ovarian suppression and preventing endogenous estradiol production while still providing highly effective , rapidly reversible , and safe contraception.713 extended - cycle levonorgestrel - ethinyl estradiol pills ( seasonale , seasonique , lo - seasonique ) belong to this newer class of ocps and offer women an 84/7 regimen with four withdrawal menses per year . lybrel is a continuous - cycle pill with active hormones taken daily throughout the year with no hormone - free interval to induce a scheduled withdrawal bleed . these four fda - approved formulations of extended - cycle ( or continuous - cycle ) levonorgestrel - ethinyl estradiol pills differ in their estrogen dosage , hormone - free intervals , and expected withdrawal bleeds ( table 1 ) . seasonale , seasonique , and lo - seasonique have varying doses of hormones and involve 3 months of continuous hormone treatment with 4 scheduled periods each year while women on lybrel ultimately experience amenorrhea after initial unscheduled spotting.14 seasonique and lo - seasonique are unique amongst extended - cycle contraceptives in offering very low amounts of estrogen during the typical 7 day hormone free interval to minimize breakthrough bleeding and decrease incidence of premenstrual symptoms.15 lo - seasonique was designed to offer women a very low dose extended - cycle ocp with theoretical decrease in estrogen - related side effects and adverse effects ( breast tenderness / nausea , vte ) though with potential for more unscheduled bleeding.14 while initial studies speculated that extended - cycle ocps may have higher efficacy rates due to the decreased likelihood of escape ovulation due to greater ovarian suppression,16,17 a systematic review of extended - cycle versus traditional 28-day cycle ocps found similar efficacy and safety . therefore , users of extended - cycle ocps can expect failure rates which vary from 0.3% with perfect use to 8% with typical use.18 as expected , users of extended - cycle ocps had greater improvement of menstrual symptoms , but despite this , the systematic review found no difference in adherence or discontinuation rates . lng is particularly popular when combined with low - dose ee in ocps because of its long track record of safety , especially when compared with third - generation progestins such as desogestrel and gestodene1921 and the newer progestin , drosperinone22,23 which have all been associated with higher rates of venous thromboembolism ( vte ) than lng - containing ocps . breakthrough bleeding is often more frequent in the extended - cycle regimens , particularly early on , but compliance rates are similar to traditional ocps . breakthrough bleeding does decrease over time with a mean number of breakthrough bleeding days of 14 in cycle one but decreasing to 8 in cycle four.15 extended - cycle ocps , particularly seasonique and lo - seasonique , may expose women to an increased cumulative amount of estrogen compared with a traditional 21/7 regimen thus making risk of vte of particular concern to patients and providers . current data does not demonstrate an increased risk of vte for extended - cycle ocps.15 additionally , there may be a concern that decreasing or eliminating withdrawal bleeding leads to endometrial hyperplasia , however studies have shown that most women have inactive or atrophic endometrium with no significant endometrial hyperplasia reported in women receiving extended - or continuous - cycle ocps.15 the risks associated with all extended - cycle ocps have been found to be similar to those of traditional ocps in a well - done systematic review , therefore it is critical to counsel women who use extended - cycle ocps on the risks and side effects associated with use of all combined hormonal contraceptives . fortunately , combination hormonal contraception can be used safely in women with a range of medical conditions including well - controlled hypertension , uncomplicated diabetes , mild hyperlipidemia , depression , uncomplicated valvular heart disease , hiv infection , various connective tissue disorders , migraines without aura , systemic lupus erythematous without antiphospholipid antibodies , and uncomplicated liver disease.24,25 prescribing ocps to healthy , non - smoking women older than 35 years old is also generally safe provided that other contraindications to combined hormonal contraception do not exist.24,26 indeed , data from us trials suggest that stroke and myocardial infarction ( mi ) risks for ocp users compared with nonusers are similar in younger and older non - smoking women.27,28 despite the varying hormonal doses and delivery methods of various ocp formulations , the risks and benefits are generally felt to be similar and are grouped together by the world health organization ( who ) . the majority of serious risks relate to the effects of estrogen on the cardiovascular system such as vte and less commonly , myocardial infarction or stroke . ocps have been shown to elevate systolic and diastolic blood pressure by about 8 and 6 mm hg respectively,29 and caution should be used in starting ocps in women who already have elevated blood pressures , especially women older than 35 years . guidelines from both the who and american college of obstetrics and gynecology ( acog ) suggest that the risks of ocps outweigh the benefits if blood pressure is uncontrolled.24,25 acog recommends that the use of ocps in women with diabetes should be limited to women less than 35 years who are nonsmoking , otherwise healthy , and show no evidence of hypertension , nephropathy , retinopathy , or other vascular disease . 24 while migraine with aura is a contraindication to the use of ocps , use can be considered for women with migraines if they do not have focal neurological signs , do not smoke , are less than 35 years of age , and are otherwise healthy.2426,30 it is important to balance contraceptive risks with the risk of complications from an unintended pregnancy in women with obesity . the who considers the benefits of ocps in obese women greater than the harms25 while acog suggests that a progestin - only method may be safer.24 the absolute risk of vte in ocp - users is generally low , and is actually lower than the risk of vte associated with pregnancy . however , the relative risk of vte in ocp - users compared to non - users is about four times higher.31 most studies suggest that vte risk is highest in the first year of use and diminishes with increasing duration of use . although combination hormonal contraception can increase risk for vte in all users , risk is especially high and is contraindicated in women with personal history of vte or with antiphospholipid antibodies.24 a complete list of contraindications for combination hormonal contraception can be seen in table 2 . lng is a more androgenic progestin which may cause some patients or providers to have concern about using extended - cycle contraceptives for those with acne or polycystic ovarian syndrome ( pcos ) , or possibly worsening these conditions . though available data is limited , similar to other formulations of combination hormonal contraception , lng decreases peripheral androgen levels.32 additionally , few differences have been seen between ocp types and effectiveness in treating acne and pcos , keeping the lng - containing extended - cycle ocps as a possible choice for women with these issues.33,34 many women still have the misconception that monthly menses remain the most natural process , therefore the newer formulations outlined above may require increased patient education on the part of providers.35 the low doses of hormones utilized in ocps do not result in buildup of the endometrium , thus no particular reason exists for a monthly withdrawal bleed beyond patient comfort . because these bleeding patterns differ largely from traditional monthly withdrawal bleeds , it s important to discuss patient preferences for menstrual frequency and tolerance for scheduled and unscheduled bleeding when deciding which contraceptive will best fit the needs of patients . nearly two - thirds of us women have demonstrated interest in menstruating less often with just over half expressing interest in menstruating every 3 months or not at all.36 it is also important to note that despite the lengthened cycles , women can expect rapid resolution of fertility after stopping extended - cycle ocps . determining a patient s preference for an extended - cycle ocp will include a discussion of the symptoms surrounding her menses , her desire to avoid menses and her ability to tolerate potential unscheduled bleeding . extended - cycle levonorgestrel contraceptives may be particularly useful for women who desire less frequent withdrawal bleeding . some studies have shown that extended - cycle ocps may be useful for women during perimenopause , with endometriosis , menorrhagia , menstrual migraine or pmdd.15 these women need to be counseled regarding the initial increased frequency of breakthrough bleeding and spotting in order to try to maximize long term compliance with their contraceptive method . the use of levonorgestrel extended - cycle ocps is safe and effective in healthy women . though they have not been compared directly to each other , each hassimilar risks and side effects to the traditional 21/7 ocp regimen . extended - cycle ocps are unique in offering fewer or no withdrawal bleeds over the course of one year but providers need to carefully counsel women regarding the initial increased breakthrough bleeding . extended - cycle ocps may be of particular benefit in women with medical comorbidities who would benefit from less withdrawal bleeds , those desiring to avoid monthly menses due to increased hormonal withdrawal symptoms , or simply women who do nt desire a monthly period .
effective contraceptive counseling requires an understanding of a woman s preferences and medical history as well as the risks , benefits , side effects , and contraindications of each contraceptive method . hormonal contraceptives using a variety of delivery methods are highly effective and this review highlights the new extended - cycle levonorgestrel - ethinyl estradiol contraceptives.extended-cycle ocps are unique in offering fewer or no withdrawal bleeds over the course of one year but providers need to carefully counsel women regarding the initial increased breakthrough bleeding . extended - cycle ocps may be of particular benefit in women with medical comorbidities who would benefit from less withdrawal bleeds , those desiring to avoid monthly menses due to increased hormonal withdrawal symptoms , or simply women who do nt desire a monthly period . the risks associated with all extended - cycle ocps have been found to be similar to those of traditional ocps therefore counseling on the risks and side effects is comparable to that of any combined hormonal contraceptives.newer extended - cycle regimens shorten or eliminate the hormone - free interval , decrease frequency of menses to four times per year or eliminate menses altogether . this can reduce the risk of common menstrual symptoms , endometriosis , or severe dysmenorrhea by offering potentially greater ovarian suppression and preventing endogenous estradiol production while still providing highly effective , rapidly reversible , and safe contraception .
You are an expert at summarizing long articles. Proceed to summarize the following text: a 52-year - old male was admitted to our hospital suffering with cough , fever and sweating for the previous two weeks . there was no history of treatment or medication for diabetes mellitus and hypertension . the patient was a smoker ( 45 pack - years ) and he drank alcohol . on the posteroanterior chest radiograph , variable sized nodules and masses were seen in both lower lobes and the right upper lobe ( fig . the contrast enhanced chest ct scan revealed variable sized masses in the right upper lobe and both lower lobes , and the masses displayed central necrosis ( fig . no endobronchial lesion was visualized on bronchoscopy . a ct - guided percutaneous transthoracic needle biopsy ( ptnb ) the histologic examination of the percutaneous transthoracic needle biopsy specimen showed diffuse infiltrates of large lymphoid cells ( fig . 1 g ) and they were negative for cd20 , cd30 ( ki-1 ) and cd56 . the specimen showed nearly normal , fully matured myeloids and an adequate number of megakaryocytes without lymphomatous involvement . 18f - fluoro-2-deoxyglucose positron emission tomography ( fdg - pet ) showed multiple hypermetabolic masses with photopenic defects ( maxsuv 8.9 ) in both lungs ( fig . finally , we diagnosed the patient as suffering with primary pulmonary peripheral t - cell lymphoma not otherwise specified . the follow - up contrast enhanced chest ct scan obtained two months after the initial diagnosis showed aggravated t - cell lymphoma involvement in the lungs ( fig . we observed multifocal irregular enhancing lesions in both parietal cortical areas on the brain ct and mri obtained four months after the initial diagnosis and this all suggested multiple parenchymal and meningeal metastases . sadly , the patient eventually expired from pneumonia during icu care after multiple episodes of neutropenic fever . primary pulmonary lymphoma is diagnosed according to the following strict criteria : 1 ) the lung , bronchus or both are involved without evidence of mediastinal adenopathy or a mass on the chest radiographs , 2 ) extrathoracic lymphoma was not previously diagnosed and 3 ) there was no evidence of extrathoracic lymphoma or lymphatic leukemia at the time that primary lymphoma of the lung was diagnosed . further , for making the diagnosis of primary pulmonary lymphoma , the disease is not present outside of the thorax for least three months after the initial diagnosis ( 2 ) . low - grade b - cell lymphoma is the most frequent form of primary pulmonary lymphoma ; however , primary pulmonary peripheral t - cell lymphoma is extremely rare . peripheral t - cell lymphoma ( ptcl ) comprises a group of rare , aggressive cancers that develop from t - cells that are at different stages of maturity . the world health organization ( who ) has divided the various types of ptcl into two main categories : 1 ) precursor t - cell neoplasms , which include precursor t - lymphoblastic lymphoma / leukemia ; and 2 ) peripheral t - cell neoplasms , which are subcategorized as predominantly leukemic disease , predominantly disseminated disease , predominantly extranodal disease and predominantly nodal disease . the cases that do not match one of the defined entities of ptcl are best categorized as ' not otherwise specified ' , reflecting the fact that we do not yet understand everything about lymphomas or the immune system ( 3 ) . the incidence of ptcl tends to be higher in korea ( 25% of the cases of nhl ) than in europe ( 10 - 15% of the cases of nhl ) , as well as the incidence of peripheral t - cell lymphoma not otherwise specified ( ptcln ) is higher in korea ( 11% of the cases of nhl ) that in europe ( 4% of the cases of nhl ) ( 3 , 4 ) . patients with ptcln are usually adults with generalized disease ; the lymph nodes , liver and spleen may be involved . the most common radiologic finding of ptcln is generalized lymphadenopathy ( 3 , 5 ) . in patients with disseminated conditions , the imaging features are not distinguishable from those of the other subtypes of lymphoma in the disseminated state . our case shows a very unique feature of primary pulmonary ptcln as the disease presented as multiple lung masses with central necrosis and there was no disease involvement at any other site on the detailed work up . the radiologic appearance of central necrosis in the consolidation or mass can also present in such benign conditions as abscess , lung infarction , c - anca ( anti - neutrophil cytoplasmic antibody)-associated granulomatous vasculitis and carcinomas . the thick wall and irregular inner margin are more frequently seen in malignant lesions , as was noted in our case . this may be due to the rapid tumor growth and it tends to occur in large nodules and masses . a cavity with air - fluid levels may be apparent when there is communication between an adjacent bronchus and a necrotic tumor mass . there has been a report about extranodal ptcl showing a pattern of photopenic defect on pet scanning , like was seen in our case ( 6 ) . this photopenic defect correlates well with central necrosis , yet necrosis is an uncommon pet scan finding for lymphoma . tumor necrosis generally correlates with hypoxia and it is a predictor of a poor prognosis for patients with malignant tumor . tumor hypoxia plays a major role in tumor progression and resistance to treatment , like what happened in our case ( 6 ) . for most of the subtypes of ptcl , the treatment regimen is typically chop - based chemotherapy ( cyclophosphamide , doxorubicin , vincristine and prednisone ) , or epoch ( etoposide added to chop ) in the frontline setting ( 7 ) . adult t - cell leukemia or lymphoma has a poor prognosis due to the life - threatening complications that include infections and hypercalcemia ( 8) . the clinical course is usually aggressive , and relapses are more common for t - cell leukemia or lymphoma than for the b - cell lymphomas of a similar histologic grade ( 9 , 10 ) . we report herein on a rare case of ptcl and we present the ct and pet images of this often fatal neoplasm .
primary pulmonary t - cell lymphoma is an extremely rare malady , and we diagnosed this in a 52-year - old male who was admitted to our hospital with cough for the previous two weeks . the chest ct demonstrated multiple variable sized mass - like consolidations with low density central necrosis in the peripheral portion of both the upper and lower lobes . positron emission tomography ( pet ) showed multiple areas of hypermetabolic fluorodeoxyglucose ( fdg ) uptake in both lungs with central metabolic defects , which correlated with central necrosis seen on ct . the histological sample showed peripheral t - cell lymphoma of the not otherwise specified form . the follow - up ct scan showed an increased extent of the multifocal consolidative lesions despite that the patient had undergone chemotherapy .
You are an expert at summarizing long articles. Proceed to summarize the following text: the incidence of thyroid cancer is dramatically rising in the unites states and other countries . thyroid cancer has increased steadily in the unites states over the past several decades , and according to data from the national cancer institute 's surveillance , epidemiology and end results ( seer ) database , incidences are now nearly three times that of the early 1970s [ 13 ] . furthermore , for unknown reasons , thyroid cancer is three times more prevalent in women than men . thyroid cancer is the sixth most common type of cancer among women and increasing more rapidly than any other cancer . the american cancer society estimates that 56,460 new cases of thyroid cancer ( 43,210 in women and 13,250 in men ) will be diagnosed in the united states in 2012 , with approximately 80% of patients below 65 years old . rates of thyroid cancer diagnoses have increased the most among small ( 2 cm ) thyroid nodules , which may be explained by the use of thyroid ultrasound for diagnosis beginning in the 1980s [ 2 , 5 ] . however , it is believed that the increase in thyroid cancer is not solely based on diagnostic methods , but due to changes in other risk factors as well . one possible contributory risk factor is exposure to ionizing radiation , either from external radiation , such as x - rays and -radiation , or internal radiation , from ingestion or inhalation of radioiodine . increases in thyroid cancer have been well documented following exposure to high doses of radiation during medical procedures or following nuclear bomb explosions or nuclear reactor fallouts . however , exposure to low doses of radiation from routine diagnostic x - ray procedures and in the workplace does not increase the risk of thyroid cancer development , suggesting radiation exposure is not the only risk factor . an increased body mass index ( bmi ) is also positively associated with thyroid cancer in both women and men , suggesting obesity is another risk factor for thyroid cancer . recently , it was observed that the chemical induction of dna breakage at chromosomal fragile sites could result in the formation of ret / ptc1 rearrangements , one common mutation observed in papillary thyroid carcinoma ( ptc ) . chromosomal fragile sites are regions of the genome prone to dna breakage and often coincide with mutations observed in cancer . exposure to a variety of external agents , including dietary , environmental , and chemotherapeutic agents , can induce breakage at fragile sites . therefore , exposure to fragile - site - inducing conditions may be an additional risk factor for thyroid cancer development , specifically ptc . interestingly , the increasing rates of thyroid cancer are almost entirely due to an increase in ptc [ 2 , 3 , 13 ] . in this paper , we will discuss the potential role of fragile sites in ptc and the external fragile - site - inducing agents that may be a risk factor for thyroid cancer . chromosomal fragile sites are nonrandom loci that can be observed as gaps or breaks on metaphase chromosomes under conditions that partially inhibit dna replication . fragile sites can further be defined as common or rare , based on the frequency of their occurrence in the population . rare fragile sites consist of repeated sequence motifs , such as trinucleotide repeats , which are present in less than 5% of the population and are inherited in a mendelian manner . in contrast , common fragile sites are present in all individuals and therefore are a normal component of chromosomal architecture . the majority of common fragile sites can be induced by aphidicolin ( aph ) , an inhibitor of dna polymerases , , and [ 17 , 18 ] . induction of other common fragile sites has been observed following treatment with bromodeoxyuridine ( brdu ) or 5-azacytidine . most rare fragile sites are expressed through the removal of folate , while others show induction following treatment with distamycin - a or brdu . additionally , common fragile site breakage can be induced or enhanced through exposure to various dietary or environmental chemicals , including chemotherapeutic agents ( details in section 4 ) . unlike rare fragile sites , however , several characteristics have been observed at many common fragile sites studied to date . these include being located within large genes and within regions of the genome that replicate late in the s - phase [ 2023 ] . also , several fragile sites contain highly flexible at - rich sequences [ 24 , 25 ] and are predicted to form stable dna secondary structures [ 24 , 26 , 27 ] . also , the atr ( ataxia - telangiectasia and rad3-related- ) dependent dna repair pathway , which responds to stalled or collapsed replication forks , is known to be vital for maintaining stability at fragile sites [ 2830 ] . one model for common fragile site breakage is that , under conditions of replication stress , stable dna secondary structures form at fragile sites , blocking replication fork progression . if the atr pathway fails to properly repair these stalled replication forks , this could result in dna breakage within these regions . in addition to replication fork stalling , paucity of replication initiation in fragile site regions and the presence of transcription - derived r - loops during dna replication of fragile sites may also be involved in the mechanism of fragility . studies over the past several decades have shown a correlation between fragile sites and cancer - specific chromosomal aberrations . many of the genes identified within fragile sites are known tumor suppressor genes or oncogenes . fragile sites have been identified as hot spots for sister chromatid exchange , viral integrations [ 3641 ] , and gene amplifications [ 4245 ] in tumor cells . additionally , mutational signatures of some unexplained homozygous deletions observed in cancer cell lines match those at fragile sites . a comprehensive examination of all known simple chromosomal translocations in cancer revealed that 52% of these recurrent translocations had at least one breakpoint located within a fragile site . specifically , 40% of translocations had breakpoints within one gene located in a fragile site , while an additional 12% of translocations had breakpoints within both genes located in fragile sites ( figure 1 ) . furthermore , 65% of the breakpoints identified within fragile sites were within common not rare fragile sites , conferring a genetic risk among all individuals . since this study only focused on simple translocations between two genes and not the participation of fragile sites in other more complex genomic rearrangements , the association of fragile sites with breakpoints in cancer may prove to be greater than estimated in this study . in addition to the correlation between fragile sites and regions of the genome mutated in cancer , two studies have investigated the direct contribution of fragile site breakage to the formation of cancer - specific chromosomal aberrations . observed deletions within the tumor suppressor gene fhit , located within the most active common fragile site fra3b , following treatment of human - mouse chromosome 3 somatic hybrid cells with aph . these deletions were consistent with those observed in esophageal , breast , and lung cancers . recently , we observed the formation of ret / ptc1 rearrangements , a translocation commonly observed in ptc , in a human thyroid epithelial cell line following treatment with the fragile - site - inducing chemicals aph , brdu , and 2-aminopurine ( 2-ap ) . for example , seckel syndrome a rare genetic disorder in which patients exhibit high levels of chromosomal instability and cancer is caused by low expression of the dna repair protein atr , due to a hypomorphic mutation in the atr gene . cells from patients with seckel syndrome have significantly higher levels of aph - induced fragile site breakage compared to normal individuals . another rare genetic disorder , fanconi anemia ( fa ) , is the result of mutations in various proteins involved in the fa double - strand dna repair pathway . chromosomal breakpoints in blood lymphocytes from fa patients are preferentially located in fragile sites , and aph - induced fragile site breakage is significantly increased among these patients . proteins in both the atr and fa dna repair pathways are important in maintaining stability at fragile sites . the wrn protein , which is phosphorylated by and colocalizes with atr in response to replication fork arrest , is also vital for maintaining common fragile site stability , and this function requires wrn helicase activity . mutations in wrn can result in werner syndrome , which is an autosomal recessive premature aging disease where individuals have a high predisposition to cancer development . interestingly , japanese werner syndrome patients have much higher levels of thyroid cancer , including ptc , than normal japanese individuals . together these provide extreme examples for a genetic predisposition for fragile site breakage and cancer development . together these previous studies provide a strong link between fragile sites and cancer , whereby exposure to fragile - site - inducing conditions and/or a genetic predisposition could attribute to the development of various types of cancer . one mutation commonly observed in ptc is ret / ptc rearrangements , in which the ret oncogene translocates with a variety of genes that are constitutively expressed in the thyroid . ret ( rearranged during transfection ) encodes for a cell membrane receptor tyrosine kinase that responds to ligands of the glial cell line - neurotropic factor ( gdnf ) family , activating cell growth and survival pathways . expression of ret in the thyroid is high in neural - crest - derived c cells but not in follicular cells , where ret / ptc rearrangements result in its activation through expression of fusion proteins leading to tumorigenesis . while prevalence of ret / ptc rearrangements is variable among different studies , overall these translocations are found in 3040% of adult and 5060% of pediatric ptc tumors . to date , 12 ret / ptc rearrangements have been reported , all involving ret . the two most common subtypes are ret / ptc1 and ret / ptc3 , where ret is translocated with ccdc6 and ncoa4 , respectively . one known risk factor for ret / ptc rearrangement formation is exposure to radiation , where the incidence of ret / ptc rearrangements in ptc patients increases to 6070% regardless of age . ret / ptc3 rearrangements have shown a strong correlation with radiation exposure , where multiple studies indicated these translocations in 6375% of radiation - induced ret / ptc - positive pediatric ptc tumors [ 6265 ] . in contrast , ret / ptc1 rearrangements have been observed in 5071% of ret / ptc - positive sporadic ptc tumors , while ret / ptc3 rearrangements were only observed in 1342% of tumors [ 62 , 6668 ] . spatial proximity of ret / ptc - participating genes is one of the factors contributing to cell specificity of the disease . the interphase distance between ret and ccdc6 is shorter in normal human thyroid cells than in peripheral blood lymphocytes or in normal mammary epithelial cells . further , ret , ccdc6 , and ncoa4 are all located on chromosome 10 and despite the predicted distance of the genes based on their location along chromosome 10 , ret is located closer to ncoa4 and ccdc6 than would be expected , during interphase in normal human thyroid cells . ret , ccdc6 , and ncoa4 , the genes participating in ret / ptc1 and ret / ptc3 rearrangements , are all located within common fragile sites . ret and ncoa4 are both located within the same aph - inducible common fragile site , fra10 g , while cccd6 is located within the brdu - induced common fragile site fra10c . the location of these genes within fragile sites , the unexplained nature of sporadic ptc tumors containing translocations of these genes , and the increasing incidence of ptc tumors suggest that fragile site breakage may contribute to sporadic ret / ptc rearrangements . the idea that fragile site breakage contributes to ret / ptc rearrangement formation was directly demonstrated in our recent publication . in this study , we first examined whether ret , ccdc6 , and ncoa4 are true fragile sites , for example , sensitive to fragile - site - inducing conditions using fluorescence in situ hybridization ( fish ) . htori-3 cells , a human thyroid epithelial cell line devoid of ret / ptc rearrangements , were treated with combinations of fragile - site - inducing chemicals known to induce the fragile sites containing the ret / ptc genes , and chromosomal breakage was measured at each gene based on the percent break of the corresponding fish probe ( figure 2(a ) ) . the presence of ret and ncoa4 in the aph - induced common fragile site fra10 g was investigated by treating htori-3 cells with aph and 2-ap . the chemical 2-ap is a general inhibitor of atr kinase , shown to increase fragile site breakage at fra3b . this treatment produced significant levels of chromosomal breakage at ret and only low levels at ncoa4 and ccdc6 , indicating that ret and not ncoa4 is contained within fra10 g . the presence of ccdc6 within the brdu - induced common fragile site fra10c was tested by treating htori-3 cells with brdu and 2-ap . high levels of breakage were observed at ccdc6 , but only low levels at ret and ncoa4 ( figure 2(a ) ) , confirming the location of ccdc6 within fra10c . these observations demonstrated that fragile - site - inducing chemicals consistent with the mode of induction for each fragile site can induce dna breakage at ret and ccdc6 in thyroid cells . furthermore , when htori-3 cells were treated with all three chemicals at once , high levels of breakage were observed within ret and ccdc6 simultaneously ( figure 2(a ) ) , indicating the possibility of ret / ptc1 chromosomal rearrangements . the major breakpoint cluster region of ret observed in ret / ptc tumor cells is intron 11 . using ligation - mediated - pcr ( lm - pcr ) , we showed that aph treatment induced dna breakage within intron 11 of ret in htori-3 cells . the rate of dna breakage at ret intron 11 with aph treatment was significantly greater than without treatment ( p = 0.010 , figure 2(b ) ) . furthermore , aph - induced breakage in htori-3 cells was specific to fragile sites , where aph treatment also induced dna breakage within fhit , located within the aph - inducible common fragile site fra3b , but not in the nonfragile 12p12.3 region or in the g6pd gene , located within the non - aph - inducible rare folate - sensitive fragile site fraxf ( figure 2(b ) ) . furthermore , the breakpoints located within intron 11 of ret were located near previously identified breakpoints in ret / ptc tumors [ 72 , 73 ] . these results suggest that fragile site breakage within ret could lead to the generation of oncogenic fusion transcripts . the generation of ret / ptc rearrangements following exposure to fragile - site - inducing chemicals was directly tested by treating htori-3 cells with aph , brdu , and 2-ap , and the presence of fusion ret / ptc1 or ret / ptc3 mrna transcripts was detected by reverse transcription - pcr ( rt - pcr ) . no ret / ptc rearrangements were detected without treatment , indicating an extremely low level of spontaneous rearrangement ( figure 2(c ) ) . however , treatment with fragile - site - inducing chemicals resulted in ret / ptc1 rearrangement events with a frequency of one in 10 cells , but no ret / ptc3 rearrangement events ( figure 2(c ) ) . these results were consistent with the chromosomal breakage observed in fish analyses , where only the ret / ptc1 genes ret and ccdc6 exhibited high levels of breakage following treatment . the data from this study provide direct evidence that fragile sites are involved in the generation of ret / ptc1 rearrangements in sporadic ptc tumors . exposure to external factors that can induce fragile site breakage may play a role in the increasing incidence of ptc . aside from classic fragile - site - inducing chemicals like aph , dna breakage at common fragile sites has been observed following exposure to many external agents , including dietary , environmental , and chemotherapeutic compounds ( table 1 ) . variability in fragile site breakage has been observed among individuals , with high levels being associated with cancer patients . these variances may reflect differing exposures to external fragile - site - inducing agents , and such exposure may predispose an individual to a variety of cancers , including ptc . numerous dietary and environmental chemicals can induce or enhance fragile site breakage ( table 1 ) . caffeine and ethanol are two dietary agents that can significantly increase the rate of fragile site breakage . caffeine , an inhibitor of phosphoinositide 3-kinase - related kinases , including atr and atm ( ataxia telangiectasia mutated ) , significantly enhances fragile site breakage in combination with aph or fluorodeoxyuridine ( fudr ) [ 76 , 77 ] . cells from chronic alcohol users have an increased frequency of fragile site breakage compared to nondrinkers , which suggests that long - term alcohol use can induce fragile site expression . exposure to cigarette smoke , pesticides , or hypoxic conditions can also increase susceptibility to fragile site breakage . peripheral blood lymphocytes from cigarette smokers have significantly greater levels of aph - induced fragile site breakage compared to nonsmokers . interestingly , peripheral blood lymphocytes from non - smokers and patients with small cell lung cancer who have stopped smoking both display lower levels of fragile site breakage following aph treatment than active smokers , suggesting this risk factor is reversible . individuals exposed to pesticides through occupational work , such as pesticide sprayers or flower collectors working in greenhouses , have increased levels of aph - induced fragile site breakage in their blood lymphocytes compared to control individuals , and these results persisted even a year later [ 8385 ] . furthermore , the pesticide - induced breakage was located within fragile sites containing breakpoints observed in non - hodgkin 's lymphoma and leukemia ; consistent with this finding , increasing rates of hematopoietic cancers have been linked to pesticide exposure [ 89 , 90 ] . hypoxic conditions also enhance fragile site breakage with or without aph treatment in cma32 chinese hamster cells . dietary and environmental agents , carbon tetrachloride , dimethyl sulfate , benzene , and diethylnitrosamine all can induce fragile site breakage . formerly , this compound was used in cleaning fluids and fire extinguishers , but has since been banned from home use due to its carcinogenic properties . an increased risk of non - hodgkin 's lymphoma has been reported among individuals working in manufacturing , industrial , and laboratory jobs in which they are exposed to carbon tetrachloride . dimethyl sulfate is used to manufacture organic chemicals including pesticides , dyes , drugs , perfumes , fabric softeners , and adhesives . dimethyl sulfate was also formerly used as a chemical weapon . occupational exposure to this compound benzene is a known human carcinogen found in gasoline , pesticides , cigarette smoke , and industrial emissions and is a common contaminant detected in food and water . according to the us department of health and human services , half of the national exposure to benzene comes from cigarette smoke , a known fragile - site - enhancing agent . the optimal concentrations of benzene ( 500 g / ml ) to induce fragile sites are relevant . smoke from a smoldering cigarette yields 345653 g of benzene , and the average exposure for one hour of driving or riding in a car is about 40 g benzene ( even greater in highly congested areas ) . therefore , the general population is exposed to a level of benzene comparable to the amount able to induce fragile sites , especially under long - term exposure . recently , pellegriti et al . observed a significantly higher prevalence of ptc in the sicilian province of catania , located near the mount etna volcano , compared to other provinces , which could not be explained by industrial pollution or mild iodine deficiencies . the authors suggest that exposure to environmental factors associated with the mount etna volcano may be responsible . benzene can form following the incomplete combustion of organic materials in volcanoes and forest fires and has been detected in lava gases emitted from mount etna , suggesting fragile site breakage due to benzene exposure may contribute to the increasing incidence of ptc in catania . furthermore , we have observed that treatment of htori-3 cells with levels of benzene previously shown to induce fragile sites results in a statistically significant increase in dna breakage within ret intron 11 , and this breakage was specific to fragile sites ( unpublished observations ) . diethylnitrosamine ( den ) is found in pesticides , cigarette smoke , industrial pollution , drinking water , and foods and beverages . den was previously used as a gasoline and lubricant additive , antioxidant , stabilizer in plastics , and in other manufacturing processes . although there are no studies relating exposure to den with cancer susceptibility in humans , many studies in laboratory animals have shown that den exposure can result in the development of various tumors . as with benzene , we found that dna breakage within ret intron 11 was significantly increased in htori-3 cells following exposure to dosages of den previously shown to induce fragile sites , and this breakage was specific to fragile sites ( unpublished observations ) . atenolol is a common -blocker used to treat hypertension , and peripheral blood lymphocytes from hypertensive patients taking this drug exhibit higher levels of chromatid and chromosomal breaks than normal individuals not taking atenolol ; these breaks were preferentially located at fragile sites . furthermore , blood lymphocytes from patients taking atenolol have significantly more micronuclei than normal patients . although antihypertensive drugs have been evaluated for carcinogenic effects , studies performed to date may not be complete enough to rule out these drugs as potential cancer - causing agents . due to the prevalent usage of hypertensive drugs and the ability of atenolol to induce fragile site breakage , usage of such drugs may be an additional risk factor for cancer development , and extensive investigations are needed . several chemotherapeutic agents are known to induce fragile site breakage including actinomycin d , bleomycin , busulfan , camptothecin , chlorambucil , cytosine arabinoside ( cytarabine ) , 5-azacytidine , floxuridine , and methotrexate ( table 1 ) [ 75 , 86 ] . the fragile site breakage observed following treatment with these chemotherapeutic agents was multiplied 3- to 8-fold by the addition of caffeine . these chemotherapeutics are commonly used to treat cancer , including leukemias and lymphomas ( table 1 ) . aside from killing cancer cells , residual doses of chemotherapy drugs can also lead to mutations in healthy cells that could result in a therapy - related second primary tumor . the plasma concentrations of cytarabine derived from the treatment dosage are comparable ( ~10 m ) or higher ( depending on the regimens ) , to the amount that causes fragile site induction ( 10 m ) . the rate of second primary cancers is on the rise , and they now account for one in six of all newly diagnosed cancers in the united states . thyroid cancer has been observed as a secondary cancer following treatment for various cancers , including hodgkin 's lymphoma [ 99 , 100 ] . patients with testicular cancer treated with chemotherapy and/or radiation also have a significantly elevated risk for developing thyroid cancer . ptc has been observed in patients treated for osteosarcoma [ 57 , 102106 ] , including children treated with chemotherapeutic agents ( some of which are known to induce fragile sites , including bleomycin , actinomycin d , and methotrexate ) , but not radiation . ptc was documented as a secondary malignancy following treatment of a pediatric rhabdomyosarcoma patient with only chemotherapeutic drugs , including actinomycin d . ptc has also been observed as a second primary cancer in children treated with chemotherapy alone for acute lymphoblastic leukemia , neuroblastoma , and ewing 's sarcoma [ 108111 ] . many chemotherapeutic agents target dna topoisomerases , acting as enzymatic poisons and resulting in an accumulation of double - strand dna breaks in cells . dna topoisomerase i activity is vital for common fragile site breakage [ 112 , 113 ] . camptothecin , one of the fragile - site - inducing chemotherapeutic agents , is a dna topoisomerase i poison . the remaining fragile - site - inducing chemotherapeutic agents perturb dna replication and/or rna transcription in cells , the mode by which many fragile - site - inducing chemicals lead to chromosomal breakage . besides the chemotherapeutic drugs already shown to induce fragile sites , many others work in a similar manner by inhibiting dna topoisomerases and perturbing dna replication or rna transcription , suggesting additional drugs may have the ability to induce fragile site breakage . together , chemotherapeutic , dietary , and environmental agents represent a diverse spectrum by which individuals can be exposed to and increase their risk of fragile site breakage . long - term exposure or exposure to significant doses of any of these agents , or a combination of different agents , the american cancer society 2012 report indicated that the incidence of thyroid cancer among women is three times higher than men . thyroid cancer is the fastest - growing cancer and the sixth most common among women . furthermore , the risk of thyroid cancer peaks earlier for women , where most women are diagnosed in the fourth and fifth decades of life compared to the sixth and seventh for men . the rate of ptc among women in the united states tripled between the early 1980s to the mid-2000s , accounting for over 75% of all thyroid cancers diagnosed during this time . despite this dramatic increase and strong disparity between women and men , however , various fragile - site - inducing factors may contribute to the difference in ptc incidence in women versus men ( figure 3 ) . the numbers of chromosomal breaks and sister chromatid exchanges are elevated in women who are pregnant or taking oral contraceptives [ 114117 ] . furthermore , women currently taking oral contraceptives have an increased incidence of thyroid cancer , including a stronger association with ptc than other subtypes . variation in the frequency of aph - induced common fragile site breakage was also observed in women during different times of the menstrual cycle , with a significant increase during the luteal phase when progesterone and estradiol levels are at their highest . estrogen and progesterone levels fluctuate throughout the menstrual cycle , but hormonal birth control prevents ovulation by maintaining consistent hormone levels using synthetic estrogens and progestins . since the development of oral hormonal contraceptives in 1960 , the rate of usage has increased , such that over 10 million women in the united states ages 15 to 44 years currently take oral contraceptives . further , 82% of reproductive - age women have taken oral hormonal contraceptive pills at some point in their life . the increasing rate of oral contraceptive use among teenage girls ages 13 to 18 is particularly striking . the percentage of women who have ever used high - dose oral contraceptives ( emergency contraception ) also rose from 4% to 10% from 2002 to 2008 . the emergency contraceptive pill plan b one - step , which contains 1.5 mg of levonorgestrel , a synthetic form of progesterone , has approximately 10-fold higher levels of the hormone than traditional contraceptives . these data , combined with the previous studies that hormone levels affect breakage within fragile sites , support a possible link between hormonal birth control use and increased fragile site breakage , but more work is needed to prove a causative role . an increasing presence of women in the workforce may also contribute to elevated incidence of ptc in women . in 1964 , only 19 million women in the united states held jobs outside of the home , compared to 65 million women in 2010 . during the 1970s and 1980s , employment in the utility , trade , and transportation industries was most popular among women , and currently these industries are ranked second . working outside of the home , including commuting to and from work , increases a woman 's exposure to environmental mutagens such as benzene and diethylnitrosamine , fragile - site - inducing chemicals found in gasoline fumes and industrial emissions . furthermore , exposure to the same amount of benzene results in approximately 20% higher levels of benzene metabolites in women versus men , indicating an increased susceptibility to fragile site breakage in women . in a study examining cancer incidence in us air force active duty personnel between 1989 and 2002 , thyroid cancer was the third most frequent invasive cancer in women and four times more prevalent than in the general population . of even greater importance , overall cancer rates among u.s . air force personnel were significantly reduced relative to the general population , suggesting occupational exposure may contribute to the difference in thyroid cancer rates . active duty air force personnel encounter unique occupational exposures that can induce fragile site breakage , including jet fuel , high altitudes , and chemical weapons . trends in cigarette smoking and alcohol consumption may also contribute to increased ptc among women . cigarette smoking has been declining in the united states over the past several decades , but the rate of decrease varies between men and women . the number of adult men that smoke decreased by 11.1% between 1965 to 2009 , but the number of women only decreased by 0.5% . nearly 66% of women aged 1844 years consume alcohol , and 14% binge drink . in 2009 , approximately 75% of high school girls reported having drank alcohol and nearly half currently consumed alcohol , both higher proportions than in their male counterparts . also , between 1979 and 2003 binge drinking among women ages 2123 dramatically increased40% among college students and 20% among nonstudents compared to a 10% decrease among men . alcohol metabolism also differs between men and women , such that when drinking the same quantity of alcohol as men , women have a higher blood alcohol content , which enhances the potential of fragile site breakage . due to the significant increase in ptc diagnoses among women in the past several decades , especially among younger women , several factors such as hormone levels , increased exposure to environmental agents at the workplace , and behaviors like cigarette usage and alcohol consumption suggest fragile site breakage may contribute to some of the increased incidence of ptc among women . the dramatic increase of thyroid cancer in the past several decades is alarming , and it is the fastest - growing cancer among women . a variety of potential risk factors for thyroid cancer , including radiation exposure , improved detection techniques , and obesity , have been proposed . chromosomal fragile sites are stable under normal conditions , but dna breakage at these sites can be induced through exposure to many external agents . the genes involved in ret / ptc1 rearrangements , ret and ccdc6 , are located within common fragile sites , and direct evidence shows that induction of fragile sites results in chromosomal breakage within these genes and ultimately leads to the formation of ret / ptc1 rearrangements . ret / ptc1 rearrangements are commonly seen in ptc tumors and are especially represented in case of sporadic ptc . therefore , exposure to external fragile - site - inducing conditions could account for some sporadic ptc tumors and may contribute to the increase in ptc incidence . various external agents can induce fragile site breakage , including dietary , environmental , and chemotherapeutic compounds . furthermore , women are especially susceptible to many of these agents , and trends over the past several decades support the involvement of fragile sites in the increased incidence of ptc in women . therefore exposure to external fragile - site - inducing agents , along with genetic factors , may predispose an individual to ptc development , making fragile sites an additional potential risk factor for thyroid cancer . further work is needed to elucidate the impact of fragile - site - inducing agents in the formation of ptc , such that the treatment of patients can be tailored and further growth in thyroid cancer incidence can be reduced .
the incidence of thyroid cancer is increasing , especially papillary thyroid carcinoma ( ptc ) , making it currently the fastest - growing cancer among women . reasons for this increase remain unclear , but several risk factors including radiation exposure and improved detection techniques have been suggested . recently , the induction of chromosomal fragile site breakage was found to result in the formation of ret / ptc1 rearrangements , a common cause of ptc . chromosomal fragile sites are regions of the genome with a high susceptibility to forming dna breaks and are often associated with cancer . exposure to a variety of external agents can induce fragile site breakage , which may account for some of the observed increase in ptc . this paper discusses the role of fragile site breakage in ptc development , external fragile site - inducing agents that may be potential risk factors for ptc , and how these factors are especially targeting women .
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Proceed to summarize the following text: computational simulation of biology , and of cancer , is an active and increasingly rich area of research.13 in in silico environments , simulation - based studies and experiments can be relatively inexpensive in cost , time , and risk , where simulations can be used as tools for hypothesis testing and predictive treatments . the trend in cancer modeling in recent years has been toward the development of multiscale models . such models allow us to capture the interdependence of biological phenomena that occur at different biological scales , for example combining models of subcellular processes with cell cell interactions , as opposed to single - scale models that might operate at one of these scales in isolation . they offer a natural framework for studying phenomena , such as cancer , which are inherently multiscale in nature , and thus appear to offer the cutting edge with regard to potential predictive power and clinical applicability . there are generally two kinds of modeling approaches4 : a bottom - up approach that looks at simulation from a functional and reductionist point of view , integrating multiple functional models of low - level processes ; and a top - down approach that is formulated from holistic observations of biological phenomena to develop models that fit observed behaviors and outcomes . in cancer modeling , both of these approaches are used to investigate and simulate different aspects of cancer , and there is an increasing interest in combining mathematical modeling techniques in a hybrid fashion . alas , as these models are typically created in isolation , interoperability is very rarely designed into the system . one of the best hopes for developing novel models of cancer that span multiple biological scales is to reuse and extend existing models . however , the integration or extension of existing models of cancer ( or elements of those models ) currently represents a substantial technical challenge in the field.5 composition of models or model components typically relies on specialist domain knowledge about model constructs , intended interactions , computational interfaces , as well as application domain knowledge , for example , the underlying biochemistry . thus , a prerequisite for composing models is being able to reason semantically about commonalities or links between different models . efforts to relate data and models to domain knowledge are common in biology as the amount and diversity of data requires standards and structures in order to effectively manage it . mature examples of open standards and ontologies include microarray and gene expression - tabular format ( mage - tab),6 biological pathway exchange ( biopax),7 and the gene ontology ( go)8,9 to name but a few . computational models can also be thought of as a kind of data , where the plethora of published models also need standards and structures . mature standards for functional descriptions of computational models include markup languages such as cellml10,11 and the systems biology markup language ( sbml).12,13 dealing with the diversity of cancer models available has been discussed previously14 and is a continuing challenge in the wider context of biological modeling when considering numerous interoperability efforts ongoing in biology , where at the time of writing there are over 545 published standards.15 the de facto technologies for linking knowledge to data lie within the semantic web stack , which includes a set of specifications and languages including the resource description framework ( rdf),16 the web ontology language ( owl),17 sparql protocol and rdf query language ( sparql),18 and the semantic web rule language ( swrl).19 these standards have been developed based on the philosophy of open data over the world wide web , and properties yielded by developing computational engines on data structures for logical inference . advanced software has been developed for logical reasoning over linked data using these standards , such as hermit20 and pellet.21 while the semantic web technology stack is mature and its standards fully supported by the world wide web consortium ( w3c ) , the primary aim of such a technology is to facilitate machine processing and interoperation in a distributed fashion across the web . the approach presented in this paper makes the assumption that all of the data , both domain - specific knowledge and model descriptions , lie within the scope of a single database , where in silico cancer models are semantically linked within this context . the overheads levied by the semantic web technology stack no longer restrict performance for querying data and for within - database analytics . our work in this paper builds on tumorml , a domain - specific xml - based markup language for computational cancer model description22 based on our experiences and requirements23 from the european commission s transatlantic tumor model repositories ( tumor ) project.24 the aim of the project was to develop a european - based digital cancer model repository to link and interoperate with a similar established digital model repository ( dmr ) based in the united states , and developed by the center for the development of a virtual tumor ( cvit).25 tumorml was developed to act as the standard communication format between elements of the tumor infrastructure , and to facilitate model exportation . its schema was designed to allow marked - up cancer model descriptions to hold essential metadata for search and retrieval of models from online repositories , as well as the linking of models via their computational interfaces . we extend and apply tumorml in this work with a property graph - based data model 26 and corresponding database implementation that semantically links model descriptions to each other via domain knowledge stored in a graph database . a property graph is a simple graph that consists of nodes and edges ( representing relationships ) , where each node and edge can possess properties that store specific values . a traversal is how you query a property graph , navigating from starting nodes to related nodes according to an underlying algorithm . in contrast to traditional relational databases , queries can be run on graph data that map more conventionally to real - world questions , as many queries deal with how entities are related rather than finding or filtering on individual properties of entities . for example , social networks are commonly expressed as graphs , where typical queries might map to questions such as who are alice s friends ? or while directed graphs have been used in data management in biology , graph models are typically used in describing biological data , such as metabolic and signaling pathways , taxonomies of terms , and structural and sequence data.2729 our approach is to combine metadata sets with representations of computational models , rather than with biological data itself . we store our graphs and queries in neo4j , an open - source graph database written in the java programming language.30 neo4j was chosen as the graph database as it is a mature , open source , general - purpose nosql database system that implements the property graph data model . neo4j uses its own query language called cypher , which was designed to be expressive of the domain at hand rather than of the data structures . they can be formulated to create and delete nodes and relationships , update properties in the graph , as well as traverse the graph and match certain sub - graph patterns . for example , given a graph consisting of nodes representing people , and relationships linking people as friend of , the cypher query to ask the above question , who are alice s friends ? , may look like that shown in listing 1 . in listing 1 , line 1 specifies to iterate through all nodes in the graph . line 2 adds a constraint as only nodes that are connected to nodes containing the property name equating to the value line 3 returns each matching node based on lines 1 and 2 . for brevity s sake , we will not describe cypher s syntax and functionality here ; a full introduction to the cypher query language can be found in a book on neo4j.31 before loading any in silico cancer models into our graph database , we define how our models should link together via some domain - specific knowledge . this provides the context for our investigation , where our semantic queries utilize domain knowledge in order to return relevant information . for example , given a model , a , we may formulate a query to ask the question , what other models are classified in the same categories as a ? in a relational model , this query would match values in a particular column to the category value , whereas in a property graph model , this query traverses the database graphs to look for models that are connected to a via a node representing the category of a. in other words , from a we traverse to a node representing the category of that cancer model , then return the set of all other model nodes connected to that category node . as a starting point , we took the data model used in the tumorml xml schema produced out of the tumor project.24 the schema allows the recording of metadata relating to cancer model descriptions on a number of levels . this enables basic curation using dublin core,32 linking with relevant people and organizations using xcard,33 as well as referencing / citation metadata using bibtexml.34 also , a tumor - specific taxonomy of cancer models was developed allowing for categorization of cancer models stored within the project s infrastructure.22 secondly , abstract model descriptions are used to describe the functional interfaces to modularized cancer models in a black box fashion where information flows through parameter descriptors . the markup is inspired by xmml.35 within the model descriptions , implementation metadata such as hardware and software environment requirements to run associated code or binary implementations are included in tumorml model descriptions . meanwhile , tables 1 and 2 summarize our mappings from the concepts outlined in figure 1 to property graph concepts . we initially loaded in three different domain- specific property graphs taken from within the tumorml schema : a tumor model metadata model ; a data model of computational types ( eg , strings , integers , etc . ) ; and a data model representing units ( si units and additional relevant units ) . figure 2 shows a visualization of our tumor model metadata model that is based on the tumor model taxonomy . what we can see here is a hierarchy where the endpoints of the graph represent taxa relating to each classifier . for example , the mathematical technique utilized , labeled math , can take one of three values continuous , discrete , or hybrid , and singlescale models may be representative of phenomena acting on specific scales , such as organ or subcellular . when new model descriptions are added to the graph database , they are categorized by connecting them to the relevant taxon . the edges connecting models to this domain - specific portion of the graph allow us to group related models together . we also load into the database domain property graphs to represent model parameter types and their units . the purpose of these is particularly relevant in the context of componentized cancer models , where linking model parameters to type and unit metadata allows us to describe how components may communicate with each other based on their computational interfaces and semantic compatibility . for example , the output of one component model may be a double - precision floating - point number , where its value represents a chemical concentration level of the phosphorolase-53 growth factor , expressed as molar concentration in m ( m = moles / liter ) . therefore , it should only be able to connect to another model via an input with the same parameter profile ( type = double - precision floating - point number ; unit = m ) . where types and units are imprecise , for example , if the unit were in nm rather than m , a computational execution environment , such as a workflow engine or simulation software , might still allow these parameters to be connected by identifying and normalizing these differences in scales . likewise , type conversions and other unit conversions could also take place . with our graph database populated with three kinds of domain property graphs ( model categorizations , computational types , units ) where in tumorml , metadata fields are used to annotate portions of models with domain - specific ontology or controlled vocabulary terms , in our graph database , we now create direct links to nodes that exist in our domain property graphs to annotate the models . our approach described so far relies on the graph database being pre - loaded with relevant domain information in order to link our cancer models . however , we can also add domain information to our domain - specific graphs on the fly by making use of external services to semantically enrich the graph database , without having to convert and load entire controlled vocabularies or ontologies into our graph database . as an example , we use ncbo s bioportal,36 an open repository of biomedical ontologies that provides access via web services and web browsers to ontologies developed in owl , rdf , obo format,37 and protg38 frames . instead of loading an entire ontology or vocabulary downloaded from bio - portal , we query bioportal for one particular term to annotate a model . in our case an appropriate bioportal query allows us to retrieve a relevant term along with its synonyms and then store these as new property graphs in neo4j . for this work , we have limited our scope to the nci thesaurus39 ( ncit ) , as it is a rich and established collection of terms with good coverage for cancer research domains , which includes community standards , such as cdisc.40 this way , models are linked via ncit codes , or by terms deemed synonymous with each other ( including abbreviations ) , allowing property graph queries to traverse between models linked by common terminology . this could be easily extended to also relate coded terms together via bioportal s user - submitted mappings that would allow graph traversals between terms taken from a variety of different vocabularies and ontologies . to illustrate how we apply our property graph model , and also how to link together models expressed as graphs , we present two examples : one for describing a single model translated from tumorml and the other for mapping a domain - specific modeling framework s modules into individual property graphs that are linked via domain - specific semantics . for our first example , we took an egfr - erk pathway module,41 which describes the ras / raf / mitogen - activated protein kinase ( mek)/extracellular - signal - regulated kinase ( erk ) pathway that is a key signaling network , governing proliferation , differentiation , and cell survival.42 briefly , binding of epidermal growth factor ( egf ) to egf receptor ( egfr ) produces a series of downstream effects through the activation of cell decision - making components.43 pathway dynamics are regulated by material balance and kinetic equations as well as by reaction rates that are dependent on the changes in concentrations of pathway components over time , as done in many other modeling studies.4446 readers are encouraged to refer to the original article to have a further understanding of the model . here , the pathway module was modeled using tumorml , as set out in previous work,22 and we transformed the tumorml description into our graph - based representation . in listing 2 , we show the tumorml description of the egfr module . in listing 3 , we show the same description expressed in a cypher query that creates relevant nodes and edges to form a sub graph . lines 1924 of the cypher query shown in listing 3 create direct links to domain - specific graph nodes , in particular to those nodes in our tumor model taxonomy depicted in figure 2 . the egfr - erk module loaded into neo4j using the cypher query in listing 3 yields the graph shown in figure 3 . when we have multiple models stored in the same database , we gradually build up connected property graphs of models clustered around metadata nodes that are part of the domain graphs . this enables us to formulate cypher queries that ask questions about the connectedness of nodes . for example , a query that returns the egfr - erk pathway depicted in figure 3 might ask the question , what models use continuous mathematics ? , where the graph database query would be what model nodes are connected to the continuous node in our database ? . line 1 of this query seeks to set the context for the pattern machine , iterating through all nodes . line 2 attempts to match sub - graphs that consist of any node , n , connected by a relationship has_metadata to a continuous node . the query then specifies to return the i d and title properties of any matching nodes as a list . a selection of matching nodes is shown in table 3 ( this is not exhaustive for brevity ) . our second example deals with angiogenesis , an essential process in normal tissue evolution and maintenance . this physiological process provides blood , which brings with it oxygen and nutrients , to many tumors allowing them to grow and spread . for many years angiogenesis has been a focus of intensive research , with several effective angiogenesis - related antitumor therapies developed by the pharmaceutical industry.47 here , we focus on a family of models developed by thomas alarcn and collaborators that has been developed into an object - oriented ( oo ) modeling framework for implementing hybrid and multiscale models of vascular tumor growth by the university of oxford s department of computer science , in conjunction with the wolfson centre for mathematical biology . the focus of the framework development has been to apply software engineering techniques that allow its elements to be highly reusable and extensible . models published by alarcn et al , in particular the family of models discussed and extended in ref . 48 , have been reverse - engineered in order to extract and abstract the common methodologies and data structures involved in the development of vascular tumor growth models . the oo framework has been developed based on these abstractions , and a functioning implementation of the framework has been developed in c++ . , we loaded into our graph database model descriptions of components of the tumor growth modeling framework described above . the reason for using such a framework for our exemplar is that it contains a coherent , modular set of models that interoperate with each other in a predictable way . thus , we are able to show how single - scale models can be linked together semantically to form multiscale models within the context of existing multiscale models . moreover , through the use of our graph - based data schema , model developers may explore the modeling framework with different kinds of queries that would not normally be straightforward without metadata and a means to compute over it . in figure 4 we have illustrated a portion of the vascular tumor growth framework , highlighting a simple case of inter facing with a model , using the unified modeling language ( uml ) where the diagram was produced using visual paradigm for uml,50 a computer - aided software engineering tool . in this case , objects of type alarcon2005subcellularmodel interact with cell objects that are collectively contained in a cellpopulation object . the parameters that are passed between the objects include chemical concentrations , and information such as cell state , mass , and cycle times . listing 5 shows the corresponding cypher query to create the property graph describing the alarcn 2005 subcellular model given in ref . now , using the example of the vascular tumor growth modeling framework , we can show how models can be linked less trivially through annotated model inputs and outputs . for example , queries involving cancer model parameters might ask , what cancer models have input parameters that are compatible with another model s output parameters ? . based on how we have linked our model parameters and variables to domain - specific data , such as using our controlled vocabulary terms , standard units , and command - line data types , we can determine the most compatible single - scale models that could be used to compose a multiscale compound model . line 1 seeks to iterate through all patterns of nodes , where the pattern consists of a model node n connected to an input parameter p , which is in turn connected to a metadata term meta that is shared with an output parameter q of a model m. next , line 2 specifies that node n should not be the same as node m ( to omit finding itself as a compatible model in the query ) . finally , line 3 specifies to return all matches as a list of pairs of model titles under two headings , where modela has outputs that match inputs for modelb , along with what terms are matched as ncit codes . , we can see that , based on the metadata terms with which model inputs and outputs are annotated , the outputs of the subcellular models are compatible with the inputs of the vegf calculator models . although the model para meters have different labels ( under table headings output a and input b ) , the annotated nci terms ensure specific and identical meaning . c1272 corresponds to the ncit code for recombinant vascular endothelial growth factor ( vegf ) , and c28217 to the term for intracellular . explicitly , then , the combination of these terms indicates that the vegf calculator components can be connected via intracellular vegf to the alarcn 2005 sub - cellular model . here , importantly , by capturing compositional relationships such as those that we know to exist between the subcellular models and vegf calculators in an existing family of multiscale models , we validate our approach . finally , another kind of query we may ask involves reasoning on compositional relationships between existing multiscale models . while the previous example uses cypher to recommend a composition , we may have stored models that we know are already composed of sub - models . such compositions are denoted in our database by the contains relationship , as shown in figure 5 . this property graph expresses the fact that the alarcn 2003 model is composed of a subcellular model , a cell proliferation model , a vascular structural adaptation model , and an oxygen calculator . each of these sub - models is associated with a specific single scale via appropriate metadata nodes . questions such as over what biological scales does the alarcon 2003 model extend ? are now possible . clearly , an appropriately phrased query would return a list containing the cell , tissue , and subcellular scales . for our first example , we took an egfr - erk pathway module,41 which describes the ras / raf / mitogen - activated protein kinase ( mek)/extracellular - signal - regulated kinase ( erk ) pathway that is a key signaling network , governing proliferation , differentiation , and cell survival.42 briefly , binding of epidermal growth factor ( egf ) to egf receptor ( egfr ) produces a series of downstream effects through the activation of cell decision - making components.43 pathway dynamics are regulated by material balance and kinetic equations as well as by reaction rates that are dependent on the changes in concentrations of pathway components over time , as done in many other modeling studies.4446 readers are encouraged to refer to the original article to have a further understanding of the model . here , the pathway module was modeled using tumorml , as set out in previous work,22 and we transformed the tumorml description into our graph - based representation . in listing 2 , we show the tumorml description of the egfr module . in listing 3 , we show the same description expressed in a cypher query that creates relevant nodes and edges to form a sub graph . lines 1924 of the cypher query shown in listing 3 create direct links to domain - specific graph nodes , in particular to those nodes in our tumor model taxonomy depicted in figure 2 . the egfr - erk module loaded into neo4j using the cypher query in listing 3 yields the graph shown in figure 3 . when we have multiple models stored in the same database , we gradually build up connected property graphs of models clustered around metadata nodes that are part of the domain graphs . this enables us to formulate cypher queries that ask questions about the connectedness of nodes . for example , a query that returns the egfr - erk pathway depicted in figure 3 might ask the question , what models use continuous mathematics ? , where the graph database query would be what model nodes are connected to the continuous node in our database ? . line 1 of this query seeks to set the context for the pattern machine , iterating through all nodes . line 2 attempts to match sub - graphs that consist of any node , n , connected by a relationship has_metadata to a continuous node . the query then specifies to return the i d and title properties of any matching nodes as a list . a selection of matching nodes is shown in table 3 ( this is not exhaustive for brevity ) . our second example deals with angiogenesis , an essential process in normal tissue evolution and maintenance . this physiological process provides blood , which brings with it oxygen and nutrients , to many tumors allowing them to grow and spread . for many years angiogenesis has been a focus of intensive research , with several effective angiogenesis - related antitumor therapies developed by the pharmaceutical industry.47 here , we focus on a family of models developed by thomas alarcn and collaborators that has been developed into an object - oriented ( oo ) modeling framework for implementing hybrid and multiscale models of vascular tumor growth by the university of oxford s department of computer science , in conjunction with the wolfson centre for mathematical biology . the focus of the framework development has been to apply software engineering techniques that allow its elements to be highly reusable and extensible . models published by alarcn et al , in particular the family of models discussed and extended in ref . 48 , have been reverse - engineered in order to extract and abstract the common methodologies and data structures involved in the development of vascular tumor growth models . the oo framework has been developed based on these abstractions , and a functioning implementation of the framework has been developed in c++ . , we loaded into our graph database model descriptions of components of the tumor growth modeling framework described above . the reason for using such a framework for our exemplar is that it contains a coherent , modular set of models that interoperate with each other in a predictable way . thus , we are able to show how single - scale models can be linked together semantically to form multiscale models within the context of existing multiscale models . moreover , through the use of our graph - based data schema , model developers may explore the modeling framework with different kinds of queries that would not normally be straightforward without metadata and a means to compute over it . in figure 4 we have illustrated a portion of the vascular tumor growth framework , highlighting a simple case of inter facing with a model , using the unified modeling language ( uml ) where the diagram was produced using visual paradigm for uml,50 a computer - aided software engineering tool . in this case , objects of type alarcon2005subcellularmodel interact with cell objects that are collectively contained in a cellpopulation object . the parameters that are passed between the objects include chemical concentrations , and information such as cell state , mass , and cycle times . listing 5 shows the corresponding cypher query to create the property graph describing the alarcn 2005 subcellular model given in ref . now , using the example of the vascular tumor growth modeling framework , we can show how models can be linked less trivially through annotated model inputs and outputs . for example , queries involving cancer model parameters might ask , what cancer models have input parameters that are compatible with another model s output parameters ? . based on how we have linked our model parameters and variables to domain - specific data , such as using our controlled vocabulary terms , standard units , and command - line data types , we can determine the most compatible single - scale models that could be used to compose a multiscale compound model . line 1 seeks to iterate through all patterns of nodes , where the pattern consists of a model node n connected to an input parameter p , which is in turn connected to a metadata term meta that is shared with an output parameter q of a model m. next , line 2 specifies that node n should not be the same as node m ( to omit finding itself as a compatible model in the query ) . finally , line 3 specifies to return all matches as a list of pairs of model titles under two headings , where modela has outputs that match inputs for modelb , along with what terms are matched as ncit codes . , we can see that , based on the metadata terms with which model inputs and outputs are annotated , the outputs of the subcellular models are compatible with the inputs of the vegf calculator models . although the model para meters have different labels ( under table headings output a and input b ) , the annotated nci terms ensure specific and identical meaning . c1272 corresponds to the ncit code for recombinant vascular endothelial growth factor ( vegf ) , and c28217 to the term for intracellular . explicitly , then , the combination of these terms indicates that the vegf calculator components can be connected via intracellular vegf to the alarcn 2005 sub - cellular model . here , importantly , by capturing compositional relationships such as those that we know to exist between the subcellular models and vegf calculators in an existing family of multiscale models , we validate our approach . finally , another kind of query we may ask involves reasoning on compositional relationships between existing multiscale models . while the previous example uses cypher to recommend a composition , we may have stored models that we know are already composed of sub - models . such compositions are denoted in our database by the contains relationship , as shown in figure 5 . this property graph expresses the fact that the alarcn 2003 model is composed of a subcellular model , a cell proliferation model , a vascular structural adaptation model , and an oxygen calculator . each of these sub - models is associated with a specific single scale via appropriate metadata nodes . questions such as over what biological scales does the alarcon 2003 model extend ? are now possible . clearly , an appropriately phrased query would return a list containing the cell , tissue , and subcellular scales . to date , many computational models of cancer have been developed to account for phenomena occurring at individual biological scales . one of the major challenges we now face is to integrate and extend these models into a fully multiscale computational framework . however , model composition and extension is non - trivial , requiring specialist domain knowledge . our approach intends to facilitate both the integration of single - scale models across multiple scales and the extension of existing multiscale models . we achieve this by enabling the annotation of cancer models and the formation of semantic links between them through the use of a property graph database . our approach , by making in silico models of cancer more understandable , also has the potential to close the gap between experimentalists and modelers and to encourage greater collaboration between them . we believe that graph databases have a very relevant place in informatics systems for cancer study , and also more broadly in biomedical informatics . while semantic technologies are mature and well supported by organizations such as the w3c , much of the philosophy behind their development is based on interoperating systems for linked data across the internet . with our tumor model graphs , we have taken the approach where metadata is stored alongside model descriptions , removing much of the overhead that comes with systems built on rdf and owl . we , however , should make it clear that we do believe that the semantic web stack has its place and its uses . for importing and exporting data and metadata from a tumor model repository , we would certainly expect to leverage some or all of these technologies , and this is why the xml markup in tumorml was originally developed to transmit such data in a standardized and interoperable format , which could be combined with the semantic web s xml - based tools and formats . in summary , we have presented a property graph model for representing tumor models so that combinations of models can be explored , based on semantic compatibility . a single graph database can be used to store domain data , such as taxonomies , controlled terminologies , and ontologies alongside model descriptions . by annotating parameters with command - line interface data types linking model descriptions to unit metadata allows us to reason about relative scalings between parameters , and by annotating with biological terms , as in the previous example using nci codes , we can check the semantic compatibility of parameters based on biological knowledge ( terms ) . we can then build relatively simple queries that propose links between models . in the future , we aim to build recommendation queries that could be quantified with compatibility metrics based on the proportions of common annotations , and also the distance of the metadata paths where terms might not match directly , but have common ancestry within ontologies or other relations between annotated terms . to date , we have demonstrated the possibilities for exploration of model composition using cypher queries and a number of existing multiscale cancer model descriptions as test data . efforts are underway to use this work as the basis for developing a set of usable software tools for exploring cancer models . in particular , we believe that the effective utilization of several modeling approaches : continuum models , discrete models as well as fitting of model parameters via biological and clinical data may be optimized using this methodology . the work described in this paper is fully available as an interactive demo and
multiscale models are commonplace in cancer modeling , where individual models acting on different biological scales are combined within a single , cohesive modeling framework . however , model composition gives rise to challenges in understanding interfaces and interactions between them . based on specific domain expertise , typically these computational models are developed by separate research groups using different methodologies , programming languages , and parameters . this paper introduces a graph - based model for semantically linking computational cancer models via domain graphs that can help us better understand and explore combinations of models spanning multiple biological scales . we take the data model encoded by tumorml , an xml - based markup language for storing cancer models in online repositories , and transpose its model description elements into a graph - based representation . by taking such an approach , we can link domain models , such as controlled vocabularies , taxonomic schemes , and ontologies , with cancer model descriptions to better understand and explore relationships between models . the union of these graphs creates a connected property graph that links cancer models by categorizations , by computational compatibility , and by semantic interoperability , yielding a framework in which opportunities for exploration and discovery of combinations of models become possible .
You are an expert at summarizing long articles. Proceed to summarize the following text: microsatellite instability ( msi ) is defined as a change in length of tandemly repeated dna sequences , caused by a failure of the dna mismatch repair ( mmr ) system to correct for such errors during dna replication . msi , the hallmark of hereditary nonpolyposis colorectal cancer ( hnpcc ) , is recognized as one of the major carcinogenetic pathways of colorectal cancer ( crc ) and is detected in 15% of sporadic colorectal cancers . crcs can be classified into 3 groups according to the msi status : msi - high ( msi - h ) , which exhibit 30 to 40% microsatellite marker instability , msi - low ( msi - l ) , which exhibit instability at < 30 to 40% of loci , and microsatellite stable ( mss ) , which exhibit no unstable markers . the primary goal of msi testing is to detect cases of hnpcc , as well as sporadic crcs developed through the replication error pathway . however , because crcs with msi - h display distinctive clinical features including proximal tumor location , poorly differentiated or mucinous histology , large size , lymphocytic infiltration , favorable prognosis , and decreased chemo - responsiveness , msi testing is also used as a prognostic marker of disease . in contrast to the strong diagnostic value associated with msi - h tumors , the biologic significance of msi - l crcs remains unclear . msi - l crcs have often been regarded as indistinct from that of mss crcs , making it uncertain whether or not msi - l crcs form a clinically unique subgroup . some studies have reported distinct clinicopathologic features associated with msi - l crcs , whereas others observed no significant differences between msi - l and mss crcs . in terms of oncologic significance , only a small number of studies examining msi - l tumors have been published , with conflicting results . to address these apparent inconsistencies , we conducted an investigation into the clinicopathologic characteristics and oncologic implications of sporadic crcs with msi - l . following review of a prospective database , we included patients subjected to surgery for sporadic crc and those with available msi results . all patients underwent radical surgery between january 2003 and december 2009 at 1 of 3 tertiary referral hospitals ( seoul national university bundang hospital , severance hospital , and samsung medical center ) , which were 1300 to 2500-bed teaching hospitals in seoul , korea . the study period was not the same among the 3 institutions , as routine msi testing with 5 microsatellite markers ( bat-25 , bat-26 , d2s123 , d5s346 , and d17s250 ) was implemented at different points of time : 2007 in seoul national university bundang hospital , 2003 in severance hospital , and 2006 in samsung medical center . however , for analyzing long - term oncologic outcomes , we enrolled patients who underwent surgery before 2009 . patients with recurrent crc , familial adenomatous polyposis , hnpcc , or inflammatory bowel disease were excluded . the basic principles of radical surgery and neoadjuvant / adjuvant therapy were similar at the 3 institutions . for clinical t34 or n12 mid - to - low rectal cancers , neoadjuvant chemoradiation was delivered ( 45.050.4 gy plus 5-fluorouracil / leucovorin ) , and radical surgery was performed 6 to 8 weeks after completion of neoadjuvant therapy . all patients underwent standard colectomy or proctectomy and regional lymphadenectomy according to tumor location , as described in our previous studies . postoperative adjuvant treatment was determined by the attending physician based on the pathologic stage and the general condition of the patient . for patients with stage ii crc with high - risk features for systemic recurrence , or stage iii crc , 5-fluorouracil - based postoperative adjuvant treatment was recommended in accordance with national comprehensive cancer network ( nccn ) guidelines . for patients who received neoadjuvant chemoradiation , postoperative adjuvant chemotherapy was recommended for all patients irrespective of surgical pathology results , in accordance with nccn guidelines . for patients with metastatic disease , cetuximab or bevacizumab all cases were restaged retrospectively according to the 7th edition of the american joint committee on cancer ( ajcc ) tnm staging system . the detailed follow - up schedule differed slightly among the institutions , although all were based on the nccn guidelines . disease - free survival ( dfs ) was determined from the date of surgery to the date of recurrence or death , with patients surviving to at least december 2014 and those lost to follow - up being censored . overall survival ( os ) was defined as the time from the date of surgery to the date of death from any cause with censoring as above . dna was extracted from paraffin - embedded tumor and surrounding normal tissues for each patient . diagnoses were confirmed via microscopic examination , with each area identified on a reference h&e - stained slide , and microdissected using a scalpel to ascertain the presence of adequate neoplastic tissue . five microsatellite markers ( bat-25 , bat-26 , d2s123 , d5s346 , and d17s250 ) were used to determine microsatellite status ; markers were selected based upon the recommendations of a national cancer institute ( nci ) workshop on msi . polymerase chain reaction ( pcr ) analyses were performed as described previously , and the shift of pcr products from tumor dna was compared to that of dna from normal colonic mucosa . tumors with at least 2 of the 5 microsatellite markers displaying shifted alleles were classified as msi - h , whereas tumors with only 1 marker exhibiting a shifted allele were classified as msi - l . samples in which all microsatellite markers displayed identical patterns in tumor and normal tissues were classified as mss . categorical variables were compared using the or fisher 's exact test ; continuous variables were compared using student 's t test . because msi - h tumors are widely known to have better prognosis , we excluded msi - h tumors in survival analyses to compare the oncologic outcome between msi - l and mss crcs exclusively . survival rates were estimated and compared using the kaplan meier method and log - rank test . cox regression was utilized for the multivariable survival analysis , and all variables with p < 0.15 on kaplan meier survival analysis were entered into the multivariable proportional hazards model . the assumption of proportional hazards was verified by examination of log cumulative hazard plots for parallelism . the predictive discrimination of each prognostic model was examined by calculating harrell 's concordance index ( c - index ) . statistical analyses were carried out using spss software version 21.0 ( ibm inc . , armonk following review of a prospective database , we included patients subjected to surgery for sporadic crc and those with available msi results . all patients underwent radical surgery between january 2003 and december 2009 at 1 of 3 tertiary referral hospitals ( seoul national university bundang hospital , severance hospital , and samsung medical center ) , which were 1300 to 2500-bed teaching hospitals in seoul , korea . the study period was not the same among the 3 institutions , as routine msi testing with 5 microsatellite markers ( bat-25 , bat-26 , d2s123 , d5s346 , and d17s250 ) was implemented at different points of time : 2007 in seoul national university bundang hospital , 2003 in severance hospital , and 2006 in samsung medical center . however , for analyzing long - term oncologic outcomes , we enrolled patients who underwent surgery before 2009 . patients with recurrent crc , familial adenomatous polyposis , hnpcc , or inflammatory bowel disease were excluded . the basic principles of radical surgery and neoadjuvant / adjuvant therapy were similar at the 3 institutions . for clinical t34 or n12 mid - to - low rectal cancers , neoadjuvant chemoradiation was delivered ( 45.050.4 gy plus 5-fluorouracil / leucovorin ) , and radical surgery was performed 6 to 8 weeks after completion of neoadjuvant therapy . all patients underwent standard colectomy or proctectomy and regional lymphadenectomy according to tumor location , as described in our previous studies . postoperative adjuvant treatment was determined by the attending physician based on the pathologic stage and the general condition of the patient . for patients with stage ii crc with high - risk features for systemic recurrence , or stage iii crc , 5-fluorouracil - based postoperative adjuvant treatment was recommended in accordance with national comprehensive cancer network ( nccn ) guidelines . for patients who received neoadjuvant chemoradiation , postoperative adjuvant chemotherapy was recommended for all patients irrespective of surgical pathology results , in accordance with nccn guidelines . for patients with metastatic disease , cetuximab or bevacizumab all cases were restaged retrospectively according to the 7th edition of the american joint committee on cancer ( ajcc ) tnm staging system . the detailed follow - up schedule differed slightly among the institutions , although all were based on the nccn guidelines . disease - free survival ( dfs ) was determined from the date of surgery to the date of recurrence or death , with patients surviving to at least december 2014 and those lost to follow - up being censored . overall survival ( os ) was defined as the time from the date of surgery to the date of death from any cause with censoring as above . dna was extracted from paraffin - embedded tumor and surrounding normal tissues for each patient . diagnoses were confirmed via microscopic examination , with each area identified on a reference h&e - stained slide , and microdissected using a scalpel to ascertain the presence of adequate neoplastic tissue . five microsatellite markers ( bat-25 , bat-26 , d2s123 , d5s346 , and d17s250 ) were used to determine microsatellite status ; markers were selected based upon the recommendations of a national cancer institute ( nci ) workshop on msi . polymerase chain reaction ( pcr ) analyses were performed as described previously , and the shift of pcr products from tumor dna was compared to that of dna from normal colonic mucosa . tumors with at least 2 of the 5 microsatellite markers displaying shifted alleles were classified as msi - h , whereas tumors with only 1 marker exhibiting a shifted allele were classified as msi - l . samples in which all microsatellite markers displayed identical patterns in tumor and normal tissues were classified as mss . categorical variables were compared using the or fisher 's exact test ; continuous variables were compared using student 's t test . because msi - h tumors are widely known to have better prognosis , we excluded msi - h tumors in survival analyses to compare the oncologic outcome between msi - l and mss crcs exclusively . survival rates were estimated and compared using the kaplan meier method and log - rank test . cox regression was utilized for the multivariable survival analysis , and all variables with p < 0.15 on kaplan meier survival analysis were entered into the multivariable proportional hazards model . the assumption of proportional hazards was verified by examination of log cumulative hazard plots for parallelism . the predictive discrimination of each prognostic model was examined by calculating harrell 's concordance index ( c - index ) . statistical analyses were carried out using spss software version 21.0 ( ibm inc . , armonk of the 3019 sporadic crc patients identified in our review , 200 ( 6.6% ) had msi - l crcs , 198 ( 6.6% ) had msi - h crcs , and the remaining 2621 ( 86.8% ) had mss crcs . msi - h tumors were associated with young age , proximal tumor location , large tumor size , poor differentiation , and n0 and m0 stages . the majority of features associated with oncologic outcome , including tumor stage , were similar between msi - l and mss ; however , some differences were observed . a statistically significant difference in tumor location was observed between msl - l and mss tumors , with 30.0% of msl - l tumors located in the right colon , compared to only 22.1% for mss msi - l tumors were also larger in their longest diameter ( mean 5.2 cm ) relative to mss tumors ( mean 4.6 cm ; p = 0.001 ) . correlation between microsatellite status and clinicopathologic variables the median follow - up time for all patients was 55 months ( range 0129 ) , with 3-year dfs seen in 83.6% of patients , and a 5-year os of 78.9% . meier survival analysis showed that preoperative carcinoembryonic antigen , tumor size , tnm stage , lymphatic , venous , perineural invasion , and tumor differentiation were associated with both dfs and os ( table 2 ) . rectal cancer was associated with shorter dfs times , whereas patient age and american society of anesthesiologists ( asa ) score were associated only with os . msi - h tumors showed better dfs ( p = 0.004 ) and os ( p < 0.001 ) compared to mss tumors ( fig . 1 ) , but msi - l was not significantly associated with dfs ( p = 0.121 ) or os ( p = 0.131 ) by univariable analysis ( fig . 1 ) ( table 2 ) . however , after adjusting for confounders via multivariable cox regression , msi - l was associated with poorer os ( hazard ratio [ hr ] = 1.354 , 95% confidence interval [ ci ] 1.0111.815 , p = 0.042 ) , as well as other factors ( table 3 ) , although it was not an independent prognostic factor for dfs ( hr = 0.704 , 95% ci 0.4621.074 , p = 0.104 ) . the c - index of each model was 0.676 for dfs and 0.805 for os . the prognostic model for os that included msi had higher c - index ( 0.805 ) than the model without msi ( 0.804 ) . univariable analysis of the prognostic factors for 3-year disease - free survival ( 3y dfs ) and 5-year overall survival ( 5y os ) kaplan meier survival curve depicting oncologic outcomes of sporadic crc patients according to microsatellite status ( msi ) . ( a ) disease - free survival . multivariable analysis of the prognostic factors for disease - free survival and overall survival figure 2 shows the distribution of unstable microsatellite markers in msi - l crcs . the majority of the 200 msi - l tumors ( 97.0% ) showed instability at dinucleotide repeats ( d5s346 13.5% [ 27/200 ] , d17s250 34.5% [ 69/200 ] , d2s123 49.0% [ 98/200 ] ) , compared with only 3.0% which displayed instability at a mononucleotide repeat ( bat26 1.0% [ 2/200 ] , bat25 2.0% [ 4/200 ] ) . . the most frequent unstable marker of msi in our msi - l tumors was d2s123 ( 49% ) . msi = microsatellite instability , msi - l = microsatellite instability - low . the analyses presented here constitute the largest study to date examining the clinicopathologic features and oncologic outcomes associated with msi - l crc . in terms of clinicopathologic features , sporadic crcs with msi - l were broadly similar to those with mss , with the only exceptions being that of tumor location and tumor size . however , among oncologic outcomes , msi - l was shown to be an independent predictor of lower os . msi - h crcs have been reported to demonstrate more frequent association with proximal location , poorly differentiated or mucinous histology , large size , favorable stage , and better prognosis , which is in consistent with our results . however , among published studies , comparisons between msi - l and mss crcs remain controversial . several studies examining crcs with msi - l were able to identify clinicopathologic features distinct from those of mss ; however , these results could not been replicated in all studies . this inherent lack of reproducibility between studies raises a fundamental question as to whether msi - l should be regarded as a distinct genetic subgroup . it is undeniable that some portion of msi - l is misdiagnosed due to technical factors including pcr amplification errors , different microsatellite markers , and cut - off levels . alternatively , msi - l may also occur as a result of random mutational events during neoplastic cell evolution . however , these hypotheses are not sufficient to explain the specificity of msi - l tumors . several lines of molecular evidence have been presented to suggest that msi - l forms a biologically discrete subgroup , including increased frequency of kras mutation , lower frequency of 5q loss of heterozygosity ( loh ) , distinct gene expression profiles in cdna microarrays , and others . here , we showed that patients with msi - l tumors had worse overall prognosis relative to those with mss tumors despite broadly similar clinicopathologic features , consistent with the notion of msi - l crcs as a distinct biological entity . future investigations using whole genome sequencing may be necessary to determine the genetic significance of msi - l , similar to what was done for msi - h tumors . beyond clinical features , a number of studies have examined the impact of msi - l on oncologic outcomes , although the results have been inconsistent ( table 4 ) . some studies described poorer survival in patients with msi - l tumors , whereas others failed to observe any significant differences or favorable oncologic outcomes as compared to patients with mss tumors . this apparent inconsistency between studies may be due to differences in study design , as each study used a different surrogate for oncologic outcome , including os , dfs , cancer - specific survival , and recurrent distant metastasis . furthermore , these studies have a number of weaknesses , such as a small number of msi - l tumors , inclusion of msi - h tumors when comparing survival outcome , or inclusion only of patients with stage ii or iii tumors . the present large multicenter study identified msi - l as an independent prognostic factor for os , consistent with previous reports of poor os in patients with msi - l tumors . clinicopathologic features and oncologic outcomes of patients with microsatellite instability - low ( msi - l ) colorectal cancers : comparison of the present study with previous studies it is noteworthy that msi - l was an independent prognostic factor for os , but not for dfs , despite the strong association generally seen between these 2 outcomes . actually , a previous study also demonstrated reduced cancer - specific survival but no difference in overall survival of msi - l compared with mss . one of the possible reasons is that significant but subtle impact of msi - l on survival might bring about this discrepancy between survival surrogates . in this case , os is considered the more definitive end point due to its obvious clinical importance and unambiguous nature , whereas dfs is not always an appropriate surrogate of os . although dfs has been shown to exhibit a strong association with os in adjuvant colon cancer studies , any statistical association between dfs and os would be weakened if survival after recurrence were prolonged . we are unable to explain why msi - l was associated with os but not dfs ; however , in this instance we think that this lack of correlation does not weaken the prognostic significance of msi - l , as the most significant association was seen with the more important survival endpoint . another statistical issue is that the prognostic effect of msi - l only emerges when using the multivariable model . frequently , a variable that is not significantly associated with an outcome by univariable analysis can be identified as an independent risk factor in multivariable analysis after adjusting for the effects of the other variables . whereas such an observation on its own may not be sufficient to prove an association , we think that the comprehensive nature of this study and the strong statistical power provided by use of a large , unbiased , multicenter study population is sufficient to demonstrate msi - l as a potential prognostic factor in sporadic crcs . previous studies have presented evidence demonstrating a distinct genetic pathway underlying the development of msi - l tumors . msi - l phenotype was reported to be strongly associated with serrated or hyperplastic polyps with an alternative histological pathway . asaka et al suggested that msi - l have different timing and frequency of the kras mutation , which may influence the oncologic outcome . some other studies demonstrated different levels of loh at 1p32 , 8p1222 , 5q , and 18q , all of which may be associated with prognosis . low expression of o - methylguanine dna methyltransferase due to promoter hypermethylation is associated with both kras and p53 mutation , which may result in poor survival . recently , some authors surmised that hypoxia associated with loss of msh3 may be related to the recurrence of msi - l tumors . further translational studies are needed to identify the distinct genetic pathways driving msi - l oncogenesis , which may provide insights into potential adjuvant therapy responsiveness , along with targeted therapy of msi - l crcs . in the present study , this observation is consistent with previous observations of dinucleotide marker instability in a majority of msi - l crcs . however , the classification of microsatellite status is , to some degree , dependent on the markers used for detection . here , we used the most widely used 5 markers recommended by the nci , although other many studies have been performed to improve the microsatellite panel for the detection of mmr defects . the revised bethesda guidelines recommended the use of 5 mononucleotide repeats for the detection of msi - h tumors due to the high sensitivity of this assay , and a simplified mononucleotide panel ( bat-25 and bat-26 ) is still used for convenience . however , using only mononucleotide markers would miss possible msi - l tumors , as they are insensitive for detecting msi - l cancers . on the other hand , the revised bethesda guidelines also recommend that a secondary mononucleotide repeat , such as bat40 , should be tested if only dinucleotide repeats are mutated . bat40 , which has been shown to be mutated in > 75% of msi - h patients , was not tested in this study , raising the possibility that some msi - h tumors may have been erroneously classified as msi - l in this study due to the panel utilized . a comprehensive microsatellite panel which includes dinucleotide markers and is sensitive for both msi - h and msi - l tumors is warranted for the accurate diagnosis on crc patients . interestingly , msi - h and msi - l tumors accounted for only 6.6% of all colorectal cancers respectively , which appears lower than that reported previously from western countries ( 1525% ) . however , the frequencies of msi - h and msi - l were not largely different among the 3 institutions ( msi - h , 3.47.4% ; msi - l , 4.17.2% ) . in the previous study , we suggested a possible explanation that ethnic differences in the molecular characteristics of colorectal carcinogenesis could affect the msi status . this large multicenter study confirmed low frequencies of msi - h and msi - l in korean patients with colorectal cancer . first , as described above , the classification of microsatellite status is limited by the number of markers used , which may have resulted in misclassifications . although the proportion of adjuvant chemotherapy was not different between msi - l and mss groups , detailed regimens , including target therapy , could have affected oncologic outcomes . lastly , because of the retrospective study design , we could not evaluate mutations of braf and kras . nevertheless , the great strength of our study is that we used large , consecutive , and multicenter study populations to reduced selection bias , and showed msi - l to be a poor prognostic factor for all stage sporadic crcs . further translational research should focus on the genetic mechanisms underlying msi - l tumorigenesis using clinically discrete groups in terms of oncologic outcome . these efforts would be the most likely to bring about a positive impact on the diagnosis and management of crcs . in conclusion , we identified msi - l as an independent prognostic factor for os in sporadic crcs despite clinicopathologic similarities between msi - l and mss tumors . further clinical and translational studies are needed to investigate the significance of msi - l in the genesis and prognosis of crcs .
abstractalthough microsatellite instability - high ( msi - h ) colorectal cancers ( crcs ) have been shown to exhibit a distinct phenotype , the clinical value of msi - low ( msi - l ) in crc remains unclear . we designed this study to examine the clinicopathologic characteristics and oncologic implications associated with msi - l crcs.we retrospectively reviewed data of crc patients from 3 tertiary referral hospitals in korea , who underwent surgical resection between january 2003 and december 2009 and had available msi testing results . msi testing was performed using the pentaplex bethesda panel . clinicopathologic features and oncologic outcomes were compared between msi - l and microsatellite stable ( mss ) crcs ; prognostic factors for survival were also examined.of the 3019 patients reviewed , 2621 ( 86.8% ) were mss , and 200 ( 6.6% ) were msi - l ; the remaining 198 ( 6.6% ) were msi - h . msi - l and mss crcs were comparable in terms of their clinicopathologic features , with the exception of proximal tumor location ( msi - l 30.0% vs mss 22.1% , p = 0.024 ) and tumor size ( msi - l 5.2 2.6 cm vs mss 4.6 2.1 cm , p = 0.001 ) . no differences were detected in either 3-year disease - free survival ( msi - l 87.2% vs mss 82.6% , p = 0.121 ) or 5-year overall survival ( os ) ( msi - l 74.2% vs mss 78.3% , p = 0.131 ) by univariable analysis . however , msi - l was an independent prognostic factor for poor os by cox regression analysis ( hazard ratio 1.358 , 95% confidence interval 1.0141.819 , p = 0.040).msi - l may be an independent prognostic factor for os in sporadic crcs despite their clinicopathologic similarity to mss . further studies investigating the significance of msi - l in the genesis and prognosis of crcs are needed .
You are an expert at summarizing long articles. Proceed to summarize the following text: the transposable element ( te ) was first described by mcclintock as a controlling element that jumps from one position to another in the maize chromosome in the mid-1940s , long before the discovery by watson and crick of the double helix structure of dna as a genetic element . it was a time when , after the rediscovery of mendel 's law of heredity in 1900 , the cytogenetic study of chromosomes was at the forefront of genetics and when genes were thought to be beads on a string located on the chromosome . it was in the late 1970s and early 1980s when insertion sequence ( is ) elements and transposons ( tn ) were discovered and found to be similar to mcclintock 's controlling elements , and her ac / ds elements were confirmed at the dna sequence level as a te . salient features of dna transposable elements ( dtes ) , including terminal inverted repeats ( tirs ) , target site duplication ( tsd ) , the transposase gene , and simple and replicative transposition mechanisms have been well established by extensive molecular biology and biochemistry studies . then , retrotransposable elements ( rtes ) , including long terminal repeat ( ltr)-retrotransposons , non - ltr - retrotransposons , and other retro - elements , were being added to the repertoire since the 1980s to make the picture diverse and complicated . many new families of tes are being added , especially from eukaryotes , even by computational screening in the post - genome era , which has necessitated a new classification based on their structures and mechanisms of transposition . the presence of foldback intercoil ( fbi ) dna was first reported by kim in 1985 and shown by a space filling model to mediate intra - molecular homologous recombination of inversions and deletions . it was further shown in 1987 that fbi dna can mediate inter - molecular dna rearrangements , such as site - specific insertions , at the foldback tip and dna transpositional integration at the intercoil end of the detached dte . it will be examined in this review how fbi dna mediation of transposition can be extended to different classes and families of rtes . readers are suggested to refer to many good reviews available on the classification , structures , genetic contents , and function of the gene products and on the mechanisms of transposition with excellent diagrammatic illustrations [ 5 , 9 , 10 , 11 , 12 ] . this review will minimize duplicate descriptions and focus on the mechanistic features that are relevant to the application of fbi dna to the mechanisms of dna transposition . to build the basis for comparison and understand transposition mechanisms , it is necessary to consider te structures based on the presence or absence of the following factors : 1 ) rna intermediates before transposition , 2 ) tirs or ltrs , 3 ) transposase or reverse transcriptase ( rt ) and other proteins related to autonomous or non - autonomous transposition , 4 ) tsd , and 5 ) other features , like dna replication modes , hairpin structure , and enhancers . transposition of dtes involves direct movement of the dte intermediate either by a simple cut - and - paste mechanism , as in tn5 , tn7 , and tn10 , or by replicative transposition , in which case one copy stays in the original site and a daughter dte copy appears in a distant location [ 16 , 17 , 18 ] . step - wise description of the transposition events includes the requirement and pairing of both dte ends [ 13 , 19 ] , binding of the transposase proteins to the terminal sequences , double - strand breaks ( dsbs ) of the dte terminals and target ends , ligation of the 3'-oh donor end and the 5'-p target end , and gap repair of the single - strand gaps on the target that were generated by dsb . in the case of the mu bacteriophage , the gap can be repaired while the entire transposon gets replicated in a cointegrate mechanism [ 21 , 22 ] . transposition of rtes involves an rna intermediate that is reverse - transcribed by rt into a cdna . this double - stranded linear dna functions as the direct precursor for integration by an integrase protein into a new location in the host chromosome in a ' copy and paste ' manner , as in the ' cut - and - paste ' transposition reaction in dtes [ 20 , 23 ] . tirs are a hallmark of dtes , as ltrs have been that of rtes until they were joined by non - ltr retrotransposons , which have a 5'-untranslated region ( 5 ' utr ) and 3 ' utr , as typified by line-1 ( l1 ) , or which has neither of the terminal elements , as typified by the alu element . it becomes clear that the presence of terminal repeats in the tes is no longer a common requirement for the transposition reaction . the description of the protein factors above will suffice for this review , except for the long interspersed nuclear element ( line ) open reading frames ( orfs ) . the retrotransposition and integration of line have been viewed as a coupled process , called target - primed reverse - transcription [ 5 , 24 ] . however , this model has recently been enhanced by the finding that l1 reverse - transcription does not require base pairing between the primer and template . therefore , the cdna that was reverse - transcribed from the mrna expressed from the genomic copy of line is inserted into the host genome . tsds are generated in almost all classes and families of tes , except for the helitron dte superfamily [ 6 , 9 , 10 ] . the size of the tsd varies from 2 to 11 , but the sequences are not conserved . the ubiquitous presence of tsds , which are generated by gap fillings ( gfs ) after te integration on the target , reflects the unity in transposition reactions , regardless of dte or rte , and the presence or absence of terminal repeats . yet , the meaning of the small oligomer size of the duplicated target is seldom discussed in terms of dna structure . helitron , a eukaryotic dte , is considered , together with polintons , a third class of tes for many unique aspects . they contain 16 - 20-bp hairpins 10 - 12 nucleotides inside of the 3'-end and transpose precisely between the 5'-a and t-3 ' with no tsd generation . helitrons indiscriminately capture and mobilize gene sequences and may impact hybrid vigor or heterosis in maize . since the hairpin sequence corresponds to the fbi dna motif , it will be interesting to consider whether it can not only serve as a terminator or rolling circle replication but also serve as a replication origin for its own replication and for frequent gene capture activities , which the helitron is capable of . polinton is 15 - 20 kb long , with a 6-bp tsd and 100 - 1,000-bp tir at both ends . polintons code up to 10 proteins , including a family of b dna polymerases ( polbs ) , a retroviral - like integrase , an a transposase , and an adenoviral - like cysteine protease . polintons , however , with their structural characteristics and presence of tsd , should follow non - homologous end - joining ( nhej ) with the gf transposition mechanism , as ordinary dtes . what makes it unique is the presence of a self - encoded polb and a short 1 - 3-bp terminal tandem repeat that supports a protein - primed self - synthesis mechanism for polinton propagation . this mechanism calls for an extrachromosomal single - stranded polinton intermediate that forms a racketlike structure and follows polb - mediated replication to become double - stranded before being integrated into the host genome . it has been proposed that eukaryotic dtes follow three types of transposition mechanisms according to different replication modes : cut - and - paste for most dtes , rolling circle replication for helitrons , and synthetic replication for polintons . in fact , there seems to be many different cases of transposon - related replication that need to be clarified in the future . as mentioned earlier , continuation of dna replication of an inserted te molecule , like in mu co - integration , may also fill the gap to generate tsds . it should be emphasized that dna transposition has been grouped in two : simple , conservative , or ' cut - and - paste ' mechanism and replicative transposition [ 16 , 17 , 29 ] . in tn5 , at the outer ends , ori - c - like 9-bp sequences are found , which may serve to direct host replication functions to the ends of the element during transposition . a synthetic mechanism is proposed for polinton propagation to explain protein - primed replication of a racket - like structure of a single - stranded polinton . if it gets to be understood that the heteroduplex structure that is derived from fbi dna may serve as the template of dna replication , many of the diverse cases of dna replication may be clarified ( see below ) . fbi dna is formed by the folding back at one point of a non - helical parallel track of double - stranded dna at as sharp as 180 and the intertwining of the bent helixes within each other 's major groove to form an intercoil with a diameter of 2.2 nm ( fig . fbi dna could be considered a double - stranded version of a hairpin or stem - and - loop structure of rna and single - stranded dna . but , such sharp bending and formation of a four - stranded intercoil are not considered possible with a double helical b - dna with a diameter of 2.0 nm . in an extended definition , fbi dna may contain a loop and a much longer stem under biological conditions . intercoil dna is designated to distinguish itself from supercoil dna , which is thicker than 2.2 nm . intercoil dna is also termed to avoid the use of four - stranded dna , which has the connotation of four - stranded base pairing among homologous repeats . it needs to be emphasized that intercoil dna is formed by two intertwining duplexes of any sequence , but when homology is met at a certain region , homologous recombination may proceed via heteroduplex formation with the help of the proper enzymes involved . fbi dna was first formulated based upon a rare but unique electron microscopy ( em ) configuration of plant mitochondrial dna ( mtdna ) molecules and space - filling models [ 7 , 8 ] . once the fbi dna structure was realized with the space filling model , it was possible to perform homologous recombination with direct contact of the repeat sequences . the intra - molecular homologous recombination of a deletion and inversion occurs via 1 ) synapsis of the repeats in the intercoil , 2 ) heteroduplex formation by 90 base flipping , and 3 ) nick - and - close resolution of the sugar phosphate backbone crossing over . inter - molecular homologous recombination , known as site - specific insertion , on the other hand , is mediated by direct contact between the attp site at the tip of the fbi dna and the attb site on the target dna . perpendicular approach of the foldback tip onto the target site effectively resolves the year - long topological puzzle encountered in four - stranded base pair winding of two circular dna molecules and explains that 6- or 7-bp attp and attb sites correspond to the diameter of the approaching fbi dna tip and the target width . dna transposition is explainable by the perpendicular approach of the detached transposon ends in intercoil form to the host target , as described in detail below . transposition of a dte molecule in fbi dna configuration is tested by a space filling model , as presented in fig . 1 and as a detailed diagram in fig . 2 . the te molecule , detached from the host by a dsb in a blunt end cut , approaches , as shown in the diagram , at a perpendicular angle to the target site . both 3'-oh ends of the te ligate with the 5'-p ends of the staggered - cut target , leaving the overhang target ends single - stranded , which would be repaired by gap filling . as shown in the diagram ( fig . 2 ) , when the te ends approach the target on the minor groove side , the target size would be 5 bp on the minor groove side and 7 bp on the major groove side . this explains the meaning of the average size of the 5 - 7-bp target duplication , as it matches the diameter of the incoming intercoil dna , 2.2 nm . in reality , the target sizes vary between 2 to even over 10 bp in a te superfamily - specific manner . this tsd size variation could be explained by several factors - namely , local squeeze and stretch of the dna double helix ; unequal cutting positions of the te ends , as exemplified in tn7 by the 3-nt overhang by staggered dsb ; the characteristics of the endonucleases involved ; and even temporary loosening of the intercoil end during a very elaborate and concerted coordination of the transpososome structure . in earlier days , when is and tn , and even the ltr retrotransposons , were the major players of transposition , it was the presence of terminal repeats that attracted attention for explaining the transpositional mechanism , as in homologous recombination . therefore , it was somewhat puzzling to explain the cases of non - ltr retrotransposons with asymmetric termini sequences . but , it is now tsd that attracts attention as a common factor for the transposition mechanism of all tes , except for helitrons . it is the structural nature of the intercoil , which is formed by intertwining duplexes , regardless of the homology between the two termini . when both ends of a te synapse in the form of an intercoil , are cut by a dsb off the host duplex strand , and approach the target in perpendicular direction , their 3'-oh ends ligate to the staggered 5'-p ends of the broken target . the resulting overhang single - strand dna gaps on both flanking ends of the target are repaired to generate the accompanying tsd . the significance of the terminal repeats is then the homologous recombination that is required for further transactions of recombination , replication , and transcription . the enhancer is a cis element of dna that affects , as a third factor , dna transactions , such as transcription , homologous recombination , and transposition [ 31 , 32 , 33 , 34 ] . it has remained somewhat of an enigma , because the presence of an enhancer element and its binding proteins at a distance in a sequence and orientation non - specific manner enhances the efficiency of the reaction by 1,000 times . rather unique configurations of rigid rod - like dna with a big protein complex at one end [ 35 , 36 fig . 4 - 2 ] may shed light on the enhancer concept and give us an idea of what actually happens under cellular conditions ( fig . 3 ) . folding back of a dna duplex at a distant point would bring an enhancer site into juxtaposition with the base of a te , which has also branched out as fbi dna ( figs . 4 and 5 ) . proteins binding to the enhancer site and to both ends of the te and any other additional factors together would form a tight dna - protein complex , called a transpososome . this transpososome would provide a tight and stable surgical platform for a series of highly concerted precision cut - and- paste reactions : namely , dsb of the te termini , sealing of the host duplexes by nhej , staggered cuts on the target site , nhej of the te 3'-oh ends and target 5'-p ends , and finally , gap - filling of the 5 - 7-nucleotide single - strand portions to flank the transposed te . in replicative transposition ( fig . first , the gap - filling reaction during tsd may continue on to the te molecule by displacement or polarized replication , even though the direction of replication may be reversible . second , as shown in the diagram , tirs in the fbi dna configuration may transform into heteroduplexes and serve as primers for bidirectional replication of the internal te , thus keeping one copy at the original site and depositing the other copy to the target site in juxtaposition . inversion of the replicated te may result from homologous recombination , as predicted [ 7 , 16 ] . tsd serves as a common landmark of unity on transposition mechanisms for both dtes and rtes . the only exception so far is the helitron superfamily , which lacks tsd and employs a rolling circle replication model . as long as a tsd is generated during te integration into the host chromosome , it indicates that the immediate intermediate of integration is in a double - stranded dna form , regardless of rna or single - stranded dna being the initial intermediate . from the em pictures of fbi and rigid rod configurations of native dna ( fig . it becomes apparent that vertical insertion of the dsb ends of the te in an intercoil alignment is probably the best - fitting mechanism of dna transposition . small sizes of the target , around 6 - 7 bp , encountered in tsd as well as in attp and attb in site - specific insertion all point to the fact that they correspond to the diameter of fbi dna 2.2 nm . the transpososome , made of fbi dna configurations of the te and the enhancer element , may effectively abolish lingering mechanistic puzzles about keeping four broken strands of the te and the host from flying apart and answer how a te molecule jumps onto a new target site at a distance . replicative transposition is explained likewise , even utilizing the terminal homology as a possible origin of replication . it would be , however , difficult , if not impossible , to reproduce such a transpososome complex in vitro by fbi dna configuration , which is critically needed for em observations and crystallography analysis . there are four main modes of transactions by which the fbi dna motif may be utilized in dna functions in the cell : alpha deletion , omega inversion , needle point site - specific insertion , and nhej - gf integration of te . with such an abundant presence in strategically important positions in the genome and very dynamic functional roles , it is hoped that fbi dna receives the proper recognition and interpretation for a better understanding of the life process .
foldback intercoil ( fbi ) dna is formed by the folding back at one point of a non - helical parallel track of double - stranded dna at as sharp as 180 and the intertwining of two double helixes within each other 's major groove to form an intercoil with a diameter of 2.2 nm . fbi dna has been suggested to mediate intra - molecular homologous recombination of a deletion and inversion . inter - molecular homologous recombination , known as site - specific insertion , on the other hand , is mediated by the direct perpendicular approach of the fbi dna tip , as the attp site , onto the target dna , as the attb site . transposition of dna transposons involves the pairing of terminal inverted repeats and 5 - 7-bp tandem target duplication . fbi dna configuration effectively explains simple as well as replicative transposition , along with the involvement of an enhancer element . the majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the fbi dna - mediated perpendicular insertion of the paired intercoil ends by non - homologous end - joining , together with gap filling . a genome - wide perspective of transposable elements in light of fbi dna is discussed .
You are an expert at summarizing long articles. Proceed to summarize the following text: penile epidermal cysts in children are usually congenital and are caused by abnormal embryologic closure of the median raphe ; these cysts are termed median raphe cysts [ 2 - 4 ] . penile epidermal cysts in adults commonly develop after trauma or surgery . during wound healing , when shedding of the epithelium occurs within a closed space , cysts are formed by squamous epithelium undergoing keratinization [ 5 - 7 ] . according to this theory , an augmentation penoplasty using a dermal fat graft can cause a penile epidermal cyst . here a 44-year - old male patient who was diagnosed with liver cirrhosis was referred to the urologic department because of a slowly growing penoscrotal swelling . the patient had a history of augmentation penoplasty with a dermal fat graft 20 years previously . the mass was recognized 4 years ago , but it was very small and asymptomatic at that time . as the swelling slowly increased in size , however , the patient had difficulty with sexual intercourse . it was round , firm , and nontender and the penile skin overlying the lesion was intact and mobile . a scrotal ultrasonography to rule out any scrotal disorder showed a 5 cm3 cm5 cm heterogeneous echoic extratesticular mass with an internal linear hypoechoic lesion with no communication with the spermatic cord ( fig . the capsule of the mass was preserved and enucleated from the surrounding tissues ( fig . histologic findings showed the mass to be a cyst lined by stratified squamous epithelium ( fig . penile epidermal cysts are broadly classified into congenital cysts and secondary cysts caused by trauma or surgery . congenital cysts , such as median raphe cysts , can arise from an abnormal embryological closure of the median raphe [ 2 - 4 ] . secondary cysts can occur because of occluded hair follicles , mechanical implantation involving the injection of epidermal fragments , and obstructed eccrine ducts [ 5 - 7 ] . because the patient in this case had undergone an augmentation penoplasty with a dermal fat graft , we suspect that the cyst originated from the dermal fat graft . amid controversies regarding the indications , the demand for and supply of augmentation penoplasty techniques in korea has increased for the improvement of personal satisfaction and self - confidence . these techniques include subcutaneous injection of fat , dermal fat grafts , autologous tissue engineering , and a fasciocutaneous flap . penile epidermal cysts can be diagnosed by careful examination and radiological evaluation , including ultrasonography and computed tomography [ 5 - 7 ] . magnetic resonance imaging may be useful in cases of suspected extension into the pelvis , although such cases are rare . if a urethral diverticula or urethrocutaneous fistula is suspected , retrograde urethrography or voiding cystourethrography should be carried out [ 5 - 7 ] . in addition , the differential diagnosis of an epidermal cyst includes teratoma and dermoid cyst . however , skin and its appendages are present in a dermoid cyst , and derivatives of other germ cells are present in a teratoma . in the present case , no appendages of skin or derivatives of other germ cells were found in the surgical specimen . the patient was misdiagnosed with a hydrocele at the time of referral . physical examination and scrotal ultrasonography surprisingly , the mass was not cystic on ultrasound , but was heterogeneous in character with an internal hypoechoic lesion . surgical excision of penile epidermal cysts is the treatment of choice [ 4 - 7 ] . although no cases of malignancy arising in the wall of an epidermal cyst of the penis have previously been reported , patients with epidermal cysts should be followed up after complete cyst removal . to our knowledge , there is little literature regarding penile epidermal cysts after augmentation penoplasty , and the incidence of cyst development related to augmentation penoplasty is also unknown . we believe the present study may be helpful to further our understanding of penile epidermal cysts after augmentation penoplasty .
a 44-year - old male patient who had undergone augmentation penoplasty 20 years previously presented with a slowly growing penoscrotal mass . the penile mass was excised totally and the pathologic diagnosis was an epidermal cyst . epidermal cysts are benign disorders that can occur in any part of the body . however , an epidermal cyst as a late complication of augmentation penoplasty is extremely rare . we report this case of a penile epidermal cyst that developed after augmentation penoplasty .
You are an expert at summarizing long articles. Proceed to summarize the following text: total tobacco cessation is the only treatment that improves the symptoms and reduces the risk of amputation in buerger 's disease . surgical revascularization is often ineffective because the distal target vessels are often involved in this diffuse segmental disease . calcium channel blockers , anticoagulants , thrombolytics , prostaglandin analogs , sympathectomy , adrenalectomy , spinal cord stimulators , omental transfers , and stem cell treatment have all been tried with limited success in decreasing rest pain and avoiding amputation . a 28-year - old male had been suffering from acute right foot pain at rest and coldness with dysesthesia , cyanosis on the right 1st toe , and claudication for 3 weeks . the ankle - brachial pressure index ( abi ) was 0.6 on the right leg . as he was a heavy smoker , the patient 's hands and feet were usually cold , and the symptoms became severe when he was exposed to a cold environment . he was free of other diseases , including autoimmune disease , hypercoagulable state , and diabetes mellitus . computerized tomography ( ct ) angiography showed segmental obstructions of the below - knee popliteal artery , which showed intraluminal thrombosis , and in the posterior tibial artery . 1 ) . we decided to operate because of the severity of the symptoms and the short segmental obstruction with thrombus . we performed endarterectomy and vein patch angioplasty of the obstructed popliteal artery with the greater saphenous vein . however , the arteriotomy site was closed with patch angioplasty because of the occurrence of a large amount of back bleeding . after the first operation , the patient 's symptoms did not improve and the patient requested reoperation to alleviate the symptoms . we performed vein patch angioplasty in the segmental obstruction of the posterior tibial artery 2 weeks after the first operation . after the operation , the patient 's symptoms improved and he was discharged on postoperative day 15 . the lumen was near - totally or totally occluded by organized thrombi that presented as recanalized vessels . remarkable chronic inflammatory cells such as lymphocytes and plasma cells were observed in the recanalized vessel wall with less inflammation in the walls of the blood vessels . ct angiography showed diffuse posterior tibial artery obstructions in the patch angioplasty of the posterior tibial artery in the upper direction ( fig . a pre - procedural angiogram showed severe stenosis in the upper part of the vein patch angioplasty of the posterior tibial artery . the possibility of stenosis and obstruction always exists in the blood vessels of patients with buerger 's disease due to inflammatory reactions . angioplasty of the posterior tibial artery was performed using a balloon catheter ( 4 - 40 mm , ever cross ; ev3 endovascular inc . , angiography after this procedure demonstrated a good final angiographic result and the absence of critical stenosis . improved blood circulation was observed in the ct angiography performed after 1 year ( fig . this patient quit smoking from before the first operation until the time of this report . for 2 months after the endovascular procedure , warfarin was used . from after the first operation until the present , aspirin and clopidogrel ( plavix ) have been used . buerger 's disease is defined as a nonatherosclerotic segmental disease that is characterized by occlusive , inflammatory , and thrombotic changes . several different criteria , such as those suggested by shionoya and olin , have been proposed for the diagnosis of buerger 's disease . typical angiographic findings were distal vessel disease with no presence of arterial wall calcification and the development of a rich typical subcutaneous network of collaterals , usually referred to as corkscrew collaterals . tobacco use , in any of its forms , plays a central role in the pathogenesis and progression of the condition . clinical presentation usually begins with ischemia of the distal small arteries and veins of the legs , arms , feet , and hands , which is manifested by claudication of the corresponding extremities . progression of the disease is typically characterized by calf claudication and , eventually , ischemic pain at rest . the prognosis for patients with buerger 's disease with respect to limb loss is significantly worse than that for patients with either atherosclerosis or the various forms of necrotizing immune arteritis . at present , the only proven strategy to prevent the progression of the disease and avoid amputation , in some cases , is the complete discontinuation of cigarette smoking or any other use of tobacco in any form . nevertheless , many patients continue to have a very poor quality of life owing to the persistence of symptoms such as intermittent claudication , raynaud 's phenomenon , or even amputations . the risk of amputation is highly correlated with continuing to smoke ; the amputation rate varies from 42% to 5% at the cleveland clinic foundation according to tobacco use . at the end of a 5-year follow - up , the median prevalence of amputation in patients with buerger 's disease , based on the most recently reported series , is reported to be as high as 24.4% for minor amputations and 8.6% for major amputations , for a total amputation rate of 33% for patients treated conservatively . surgical revascularization for patients with buerger 's disease is possible only in a few cases , as a result of diffuse distal involvement with no distal runoff vessels available for bypass surgery . consequently , surgical procedures are technically challenging , with very low feasibility and patency rates . endovascular treatment for buerger 's disease is not generally reported in the medical literature as either feasible or effective . graziani et al . reported that an endovascular procedure is safe , technically feasible , and effective , independent of the continued or non - use of tobacco , which leads us to argue that the procedure alone achieved these high rates of clinical success , the decreased rates of amputation , and the maintenance of clinical improvement during midterm follow - up periods . although the physiopathology of the disease is characterized by the presence of inflammation arteritis and associated thrombosis , this did not preclude or affect the results of the primary angioplasty of the treated lesions . in our case , the patient was suspected of having a subacute form of buerger 's disease on the basis of the microscopic findings . it seems that inflammatory changes in blood vessel obstruction were in progress after the patient underwent patch angioplasty of the segmental obstruction . we resolved this by endovascular intervention , and we have been observing the patient 's progress for about a year , but no strictures or other abnormalities have been found . finally , although tobacco cessation is one of the most important steps for a significant reduction in disease progression , we believe that an endovascular procedure can be an effective treatment , even in addition to more conservative and surgical management , in patients with buerger 's disease and critical limb ischemia .
surgical revascularization for patients with buerger 's disease is possible only in a few cases , due to the diffuse segmental involvement and the lack of distal runoff vessels available for bypass surgery . we encountered a case of resting pain in the right foot , coldness with dysesthesia , and cyanosis on the right 1st toe . the patient was treated with an endovascular intervention after vein patch angioplasty failed due to an inflammatory reaction of buerger 's disease . we suggest that an endovascular procedure can be an effective treatment , even in addition to more conservative and surgical management , in patients with buerger 's disease and critical limb ischemia .
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Proceed to summarize the following text: ectopic pregnancy occurs in 1.5%2% of all pregnancies.1 four different management strategies for ectopic pregnancy may be considered : expectant management ( follow - up until decrease of human chorionic gonadotropin [ hcg ] rate ) , medical treatment , conservative surgery , and radical surgery . nonetheless , care for women with ectopic pregnancies remains a topic of debate , and no consensus or guidelines exist to clarify the choice between different treatments.2 activity of ectopic pregnancy ( allowing to differentiate between ectopic pregnancies that can benefit from a medical treatment and those requiring a surgical management ) is probably the major concept to decide the most appropriate treatment.3 this concept is well recognized and generally applied to determine which ectopic pregnancies could benefit from medical treatment . nonetheless , the definition of activity is also not consensual ; decision criteria are different from one study to another . even for initial rate of hcg , the decision thresholds are not the same between studies . earlier diagnosis and better access to care leads to the possibility of medical treatment in many cases . medical management for ectopic pregnancy is subject to substantial variations with different protocols and various routes of administration . regardless the protocol used , methotrexate is currently the medical treatment of choice for ectopic pregnancy . finally , the use of surgical management to recover is needed in 10%25% of cases after medical treatment with methotrexate.48 this risk of a rescue surgery is a main concern . recently , some studies suggested combining gefitinib and methotrexate to improve medical treatment and to decrease the need for reinjection and additional surgery.9,10 the aim of this review is to evaluate efficacy and safety of gefitinib in combination with methotrexate in different indications such as tubal and non - tubal ectopic pregnancy and also to report tolerance of this combination , the need for additional injection of methotrexate , and rates of additional surgery . tanaka et al reported the first use of methotrexate as medical treatment for ectopic pregnancy in 1982.11 methotrexate is an antimetabolite that acts by actively proliferating cells , including trophoblastic tissue . the dose of methotrexate used in ectopic pregnancy is 1 mg / kg or 50 mg / m . this treatment can be injected intramuscularly or directly into the ectopic pregnancy under sonographic guidance.12 some protocols advocate the use of a single injection with rescue injection if necessary , whereas other protocols recommend the use of two , three , or four systematic injections every other day.7,13,14 there are no studies comparing all these protocols for efficacy , rate of additional surgery , and safety ( rate of side effects ) . barnhart et al compared single dose and multi - dose protocols and concluded that single dose a comparison to a two - dose regimen should be done.15 in all published studies , the rate of additional surgery to recover after methotrexate injection is 10%25%.48 reported side effects are minor and transient , mainly abdominal pain , asymptomatic elevation of liver enzyme , and in rare cases ( approximately 1% ) transient hepatitis has been reported.6 gefitinib is an orally administered egf ( epidermal growth factor ) receptor - tyrosine kinase inhibitor used in breast cancer and non - small - cell lung cancer.1618 it is a drug that selectively inhibits the tyrosine kinase domain of the egf receptor and blocks signal transduction pathways implicated in the proliferation and survival of cancer cells.19 normal human placenta expresses very high levels of egf receptor , more than 30 times higher than average tissue expression . it has the highest expression of egf receptor compared to all other non - malignant tissues.20 the presence of receptors in the placenta of an ectopic pregnancy has also been confirmed21 specifically on the outer membrane of the syncytiotrophoblast . preclinical studies were performed to evaluate combination of methotrexate and gefitinib compared to methotrexate alone and gefitinib alone for inhibition of placental cell growth . inhibition of placental cell growth by methotrexate alone is dose dependent and gefitinib alone had little effect on placental cell growth . this study also demonstrated that combination of methotrexate and gefitinib not only inhibited cell growth but also induced cell death.21 in in vivo studies on mice with subcutaneous human placental cells on one hand and intrauterine early pregnancy on the other hand , a dose dependent response to methotrexate alone was observed similarly to gefitinib alone , but not with in vitro models . the use of both treatments together significantly increased response compared to methotrexate alone or to gefitinib alone.21 twelve women with ectopic pregnancy were included in a phase i study treated with methotrexate ( 50 mg / m , intramuscularly ) and gefitinib ( 250 mg orally on day 1 for three women , three times on day 1 , 2 , and 3 for three women , and seven times on day 1 , 2 , 3 , 4 , 5 , 6 , and 7 for six women ) . ten out of 12 women s ( 83% ) ectopic pregnancies resolved with the combination of methotrexate and gefitinib and one out of ten needed a rescue methotrexate injection . compared to 71 historic controls treated with methotrexate alone , hcg levels by day 4 and by day 7 after treatment among women treated with combination treatment were significantly lower ( p<0.05 ) and median recovery time was 11 days shorter in this group ( p=0.02).10 a phase ii open multi - center clinical trial has been conducted to evaluate efficacy of a combination of gefitinib ( 250 mg orally , daily for 7 days ) and methotrexate ( 50 mg / m , intramuscularly ) in the treatment of ectopic pregnancy . the study protocol was published in 2013 ; results should be readily available.9 for non - tubal ectopic pregnancy , horne et al published a case series of non - tubal ectopic pregnancies treated with methotrexate and gefitinib . eight women were included in this study , five with interstitial ectopic pregnancies and three with cesarean scar ectopic pregnancies . no surgical treatment was required and five women out of eight required a second methotrexate injection ( three of them because of a significant rise in serum hcg between days 1 and 4 ; the two others because serum hcg had not fallen more than 15% between days 4 and 7).22 in a phase i study comparing 12 women treated with the combination of methotrexate and gefitinib to 71 historic controls treated with methotrexate alone , toxicity reported with combination treatment was acneiform rash in 67% of cases and diarrhea in 42%.10 they were always transient and are known side effects of gefitinib previously described in lung cancer.23 no clinical or biochemical evidence of serious side effects ( eg , renal , hepatic , pulmonary , or hematologic toxicity ) was recorded.10 horne et al have reported the same side effects in addition to dizziness with combination of methotrexate and gefitinib in non - tubal ectopic pregnancy.22 these results have yet to be confirmed in a phase ii trial ; the protocol of such study has already been published , results are expected.9 these preliminary results are very promising but need to be explored further before wide distribution . however , if efficacy of medical treatment improves with this combination , indications could expand . the major point of interest of this combination is the increase in success of medical treatment alone , the diminution of rescue injection of methotrexate , the reduction of healing time , and the cost saving that could be generated . reduction in healing time avoids the need for repeat hcg blood samples until recovery ; increased success of medical treatment alone limits hospitalization and surgery ; and diminution of rescue injection of methotrexate also reduces costs . other points should be explored such as evaluation of combination of in situ methotrexate and gefitinib . a retrospective study suggests a superiority of intramuscular route.12 this route might also be enhanced by gefitinib . systematic postoperative injection of methotrexate has also been debated because of its efficacy weighed against the risk of side effects.2426 as gefitinib seems to be well tolerated , it should be interesting to use it after conservative surgery ( salpingotomy ) to avoid persistent trophoblasts . another real challenge is to improve medical management of non - tubal ectopic pregnancy , which presents an important hemorrhagic risk . the preliminary results of horne et al about the use of methotrexate and gefitinib in the management of non - tubal ectopic pregnancy are very encouraging and should be developed to enable use in clinical practice.22 rates of subsequent fertility after medical management and conservative surgery seem to be similar according to a randomized trial,6 but the combination of methotrexate and gefitinib should not only be evaluated comparing medical treatment with methotrexate but also with conservative surgery . finally , choriocarcinoma might be another medical condition that could be treated with the combination of both drugs . combination of gefitinib and methotrexate might be the future for medical treatment of ectopic pregnancy . however , phase ii and phase iii studies have to be conducted before large utilization . for other indications , such as non - tubal ectopic pregnancy or choriocarcinoma ,
medical management for ectopic pregnancy is subject to substantial variations with different protocols and various routes of administration . regardless the protocol used , methotrexate is currently the medical treatment of choice for ectopic pregnancy . the risk of a rescue surgery is a main concern . recently , some studies suggested combining gefitinib and methotrexate to improve medical treatment and to decrease the need for reinjection and for additional surgery . gefitinib is an orally administered egf receptor - tyrosine kinase inhibitor . for tubal ectopic pregnancy , median recovery time was shorter after combination treatment with gefitinib and methotrexate . toxicity reported with combination treatment was acneiform rash in 67% of cases and diarrhea in 42% . they were always transient and are known side effects of gefitinib previously described in lung cancer . these preliminary results are very promising but need to be explored further before wide distribution . for ectopic pregnancy , combining treatment seems to be interesting but results of the first randomized trial have to be evaluated first . for other indications , such as non - tubal ectopic pregnancy or choriocarcinoma , randomized studies are needed before wide use of the combination in current practice .
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Proceed to summarize the following text: apoptosis , also known as the programmed cell death , is a biological process that regulates physiological cell death and plays an important role in the pathogenesis of a variety of human diseases , including cancer . resistance to apoptosis or reduced cellular apoptotic capacity ( ac ) provides a survival advantage of the cells that may develop into cancer cells , commonly seen in almost all types of malignant diseases , and mutations in the genes involved in apoptotic pathways are one of the molecular mechanisms underlying carcinogenesis [ 2 , 3 ] and cancer therapy [ 3 , 4 ] . benzo[a]pyrene ( b[a]p ) is a classic dna - damaging carcinogen found in both tobacco smoke and the environment as a result of fuel combustion . its bioactivated form , benzo[a]pyrene diol epoxide ( bpde ) , can cause irreversible damage to dna by forming dna adducts through covalent binding or oxidation [ 6 , 7 ] . if these adducts are unrepaired , the cells will undergo apoptosis through activation of p53 , caspase-9 ( casp9 ) , and caspase-3 ( casp3 ) [ 8 , 9 ] . as a pivotal regulator of cellular response to dna damage , the transcription factor encoded by the p53 tumor suppressor gene has been clearly implicated in b[a]p - induced apoptosis , and the levels of p53 protein expression has been correlated with the levels of b[a]p - dna adducts [ 8 , 10 ] . although details of the signaling pathways that trigger apoptosis in lymphocytes remain not fully understood , possible mechanisms include transcriptional activation of the bcl-2 family members and transcriptional upregulation of the death receptors ( drs ) [ 12 , 13 ] . these complex proteins participate in the activation of a sequential signaling that modulates two main apoptotic pathways . one is the intrinsic or mitochondrial pathway , in which the stimuli of p53-bcl-2 pathway lead to the activation of casp9 and release of cytochrome c from the mitochondria . the other , referred to as the extrinsicor cytoplasmic pathway , involves a group of proteins such as the drs , the membrane - bound fas ligand , the fas complexes , the fas - associated death domain , caspase-8 ( casp8 ) , and caspase-10 ( casp10 ) [ 15 , 16 ] . activation of these two pathways initiates a common downstream proteolytic cascade that involves casp3 and caspase-7 ( casp7 ) . therefore , we hypothesized that functional polymorphisms in genes involved in these apoptotic pathways may modulate the ac phenotype , thus contributing to individual variation in response to dna damage . to test this hypothesis , we selected 14 potentially functional polymorphisms in 11 genes , that is , p53 , bcl-2 , bax , and casp9 involved in the intrinsic pathway ; dr4 , fas , fasl , casp8 , and casp10 involved in the extrinsic pathway ; and casp3 and casp7 , the effective casps . we genotyped for these 14 polymorphisms and assessed in vitro ac with a terminal deoxynucleotidyl transferase - mediated dutp - biotin nick - end labeling ( tunel ) assay using bpde - treated primary lymphocytes from 172 subjects without cancer to evaluate associations between their apoptotic genotypes and the ac phenotype . subjects in the current study were the control subjects in a molecular epidemiology study of lung cancer previously described . briefly , 172 subjects in this study were randomly selected from a pool of cancer - free control subjects recruited from the kelsey - seybold clinics , a large multispecialty physician organization with several clinics throughout the houston metropolitan area . each subject was scheduled to be interviewed after a written informed consent was obtained . after the interview , a venous blood sample of about 20 ml was collected from each subject . the research protocol was approved by the university of texas m. d. anderson cancer center and the kelsey - seybold foundation institutional review boards . we used the national center for biotechnology information ( ncbi ) dbsnp database ( http://www.ncbi.nlm.nih.gov ) , the national institute of environmental health sciences ( niehs ) environment genome project snp databases ( http://egp.gs.washington.edu/directory.html and http://www.genome.utah.edu/genesnps/ ) and literature search to identify potentially functional variants in genes involved in both intrinsic and extrinsic apoptotic pathways . polymorphisms with a minor allele frequency ( maf ) of 0.05 were included , if they may theoretically result in amino acid changes ( nonsynonymous snp , nssnp ) , located at regulating regions such as promoters , or are reportedly associated with known phenotypic effects . three reported p53 snps were selected , including the well - known codon 72 snp ( r72p , g > c ) and two intronic variants ( a 16 bp - del / ins in intron 3 and a g - to - a transition in intron 6 because their haplotypes were found to be functional . two previously reported regulating snps in the promoters of the bcl-2 family members , bcl-2 ( 938c > a ) and bax ( 248 g > a ) [ 19 , 20 ] , were included . for the casps , we identified one nssnp from each casp8 ( d302h , g > c ) , casp10 ( i522l , a > t ) , and casp7 ( d255e , c > g , http://www.ncbi.nlm.nih.gov ) and one of the two casp9 nssnps in tight linkage disequilibrium ( ld ) ( q221r , g > a , http://egp.gs.washington.edu/directory.html and . because no nssnp was found in the coding region of casp3 , we selected one common variant in its promoter region : 1337c > g ( http://www.genome.utah.edu/genesnps/ ) . for the death receptor genes , one nssnp in dr4 and three promoter snps in fas and fasl were selected : t209r ( c > g ) in dr4 , 1377 g > a and 670a > g in fas , and 844 t > c in fasl [ 2326 ] . the genotyping methods used to distinguish the 14 selected polymorphisms in 11 apoptosis - related genes are presented in table 1 . genotyping methods for seven of the polymorphisms were previously described : p53 r72p ) , p53 intron 3 16-bpdel / ins and intron 6 g > a , dr4 t209r , fas 1377 g > a and 670a > g , and fasl 844 t > c . the remaining seven polymorphisms ( i.e. , bcl-2938c > a , bax248 g > a , casp9 q221r , casp8 d302h , casp10 i522l , casp3 1337c > g , and casp7 d255e ) were detected by using a primer - introduced restriction analysis ( pira)polymerase chain reaction ( pcr ) assay and summarized in table 1 . genotyping was performed without knowledge of the subjects ' phenotype ; more than 10% of the samples were randomly selected for confirmation , and the results were 100% concordant . for the seven self - designed genotyping assays , pcr products containing each target genotype were purified and the sequences were confirmed by direct sequencing . the apoptosis phenotype ( i.e. , apoptotic capacity [ ac ] ) was detected with the tunel assay previously described . briefly , two parallel short - term cultures from each blood sample were incubated at 37c without co2 for 67 hours before bpde treatment . at the end of the incubation , one of the two parallel cultures was treated with bpde ( 98% pure ; midwest research institute , kansas city , mo , usa ) at a final concentration of 4 m . after an additional 5-hour incubation , all cells were pelleted by centrifugation , resuspended with lysis buffer ( human erythrocyte lysing kit , r&d systems , minneapolis , minn , usa ) , fixed for 1 hour , rinsed with phosphate - buffered saline , and finally stored in 70% ethanol at 20c until used for the tunel assay . for the tunel assay , we used the apo - brdu kit ( phoenix flow systems , san diego , calif , usa ) and followed the manufacturer 's recommended protocol . the ratio of the difference in the percentages of apoptotic cells in a subject 's bpde - treated and untreated cultures to the percentage of apoptotic cells in the untreated culture was recorded as the ac ( ac% = [ actreated - acbaseline)/acbaseline ] 100 ) . dna quality or quantity was insufficient for genotyping in 2 subjects ; thus , the final analysis included 170 persons . differences of the continuous ac measurements between genotypes / diplotypes of apoptotic genes were evaluated by using student 's t - test . trend test was performed by using the general linear regression model with adjustment for age and sex . we dichotomized the continuous phenotype measurements by using the median ( 150% ) as the cutoff value to obtain an almost equal low - ac subgroup ( 84 subjects ) and high - ac subgroup ( 86 subjects ) . logistic regression analyses were used to estimate theodds ratios ( ors ) and 95% confidence intervals ( cis ) between combined genetic variants and dichotomized ac phenotype with adjustment for age and sex . alleles / haplotypes associated with the lower ac phenotype in individual polymorphism analysis were termed as at - risk we used the phase 2.0 program to infer haplotype frequencies based on the observed genotypes for each gene . the potential gene - gene interaction was evaluated by logistic regression analysis and tested by comparing the changes in deviance ( 2 log likelihood ) between the models of main effects with or without the interaction term . all of the statistical analyses were performed with statistical analysis system software ( v.9.1.3 ; sas institute , inc . , cary , nc , usa ) . the mean age ( sd , years ) of the 170 study subjects ( 119 males and 51 females ) was 57.99 12.10 , and we dichotomized age by using the cutoff value of 60 years to facilitate comparisons between age groups . no statistical difference was found in the continuous ac measurements between the subgroups according to age and sex ( data not shown ) . table 2 shows the continuous ac measurements by genotypes of the selected apoptotic genes . the observed genotype frequencies were all consistent with those expected from the hardy - weinberg equilibrium ( data not shown ) . for snps in genes involved in the intrinsic apoptotic pathway , variant homozygotes of p53 intron 3 16-bpins / ins , p53 intron 6 aa , and bcl-2 938aa all had significantly higher ac than their corresponding wild - type homozygotes ( 496.07 121.26 versus 204.22 183.21 for p53 intron 3 16-bpdel / ins , p = .027 ; 496.07 121.26 versus 199.44 179.10 for p53 intron 6 g > a , p = .021 , and 247.62 225.67 versus 164.06 154.89 for bcl-2938c > a , p = .046 ) . however , the significant p values for the trend of higher ac with increasing number of the variant alleles were observed only for p53 r72p ( .016 ) and bcl-2 938c > a ( .037 ) as assessed in the general linear regression model with adjustment for age and sex ( table 2 ) . in contrast , only the variant homozygotes of casp10 i522l out of all snps in genes involved in the extrinsic apoptotic pathway had significantly lower ac ( 159.49 171.44 ) than the ii homozygote ( 239.07 205.18 , p = .046 ) as well as a significant trend of lower ac with increasing number of the variant alleles ( p = .046 ) . linkage disequilibrium ( ld ) analysis showed that the three loci in p53 were in ld ( r = 0.14 , d = 0.64 for r72p and intron 3 16-bpdel / ins ; r = 0.24 , d = 0.85 for r72p and intron 6 g > a ; and r = 0.60 , d = 0.79 for intron 3 16-bpdel / ins and intron 6 g > a ) . therefore , we performed haplotype / diplotype inference using the phase 2.0 program based on the observed genotypes . the diplotype carrying zero copy of the p53 r - del - g haplotype and the diplotype carrying two copies of the p53 p - ins - a haplotype all had significantly higher ac ( termed as protective hereinafter ) and the effect of the r - del - g haplotype was in a dose - response manner ( p for trend : .016 ; table 3 ) . in the genotype - phenotype analysis , we examined the role of potentially functional variants in selected apoptotic genes in the ac phenotype induced by bpde treatment in primary lymphocytes . we found that r72p , intron 3 16-bpdel / ins , intron 6 g > a in p53 , 938c > a in bcl-2 , and i522l in casp10 may be predictors of ac , but the effects of the p53 variants might also be modulated by its downstream genes involved in the intrinsic pathway . to the best of our knowledge , this is the first multigene genotype - phenotype correlation analysis in relation to the apoptotic pathways in primary lymphocytes at a population level . because there is tissue specificity in response to carcinogen exposure , it would be ideal to compare the bpde - induced ac measurements among different tissues of the same person . however , few reported studies have addressed this tissue specificity , nor did our study have such an opportunity . it was reported that b[a]p - induced apoptosis of murine hepa1c1c7 cells was through casp-9 activation related with p53 accumulation and activation and that a decrease in the expression of bcl-2 to bax ratio was another hallmark of the process [ 8 , 34 ] . although obtained from different cell types , these findings are consistent with our current observations that genetic variants in the genes involved in the intrinsic apoptotic pathway may play an important role in the prediction of ac phenotype . the bcl-2 family is a group of evolutionarily conserved pro- and antiapoptotic proteins that play a pivotal role in the regulation of the mitochondrial - mediated ( intrinsic ) apoptotic pathway . bcl-2 inhibits apoptosis through heterodimerization with proapoptotic members of the bcl-2 family , such as bax and also through formation of channels that stabilize the mitochondrial membrane . bcl-2 expression has also been implicated in the pathogenesis of cancers [ 37 , 38 ] , and the expression of bcl-2 to bax ratio seems to be important in determining both in vitro and in vivo response to chemotherapeutic drugs . recently , variant allele of bcl-2 938c > a was found to be associated with reduced prostate cancer risk in caucasians in a small case - control study , possibly due to the elimination of an sp1 binding site , a downregulation of bcl-2 mrna transcript levels , and unregulated programmed cell death , which is consistent with what we found in the current study ( the variant a allele carriers were associated with high - ac phenotype that may help eliminating possible malignant cells ) . for example , the wild - type 72r allele was found to be associated with an increased ability to induce apoptosis in response to radiation or cytotoxic drugs [ 18 , 41 , 42 ] . however , other data suggested that the 72p allele had a stronger transcription effect in response to dna damage , leading to enhanced apoptotic phenotype . these previous studies were mainly based on assays with cell lines [ 18 , 41 ] and suffered with a limited sample size [ 18 , 41 , 42 ] . most importantly , no previous study took into account other coexisting polymorphisms in the genes involved in the intrinsic pathway , which may play an important role in the b[a]p - induced apoptosis . in the present study with apparently normal primary lymphocytes , we found that all minor variant alleles of the three p53 polymorphisms were associated with a higher ac phenotype and that the overall effects on the ac phenotype appeared to be affected by the casp9 q221r polymorphism , but the interaction between polymorphisms of p53 and casp9 was only borderline significant due to a limited study power . however , our findings of this intrinsic apoptotic pathway in lymphocytes may be relevant to other types of tissue as well because this kind of induced apoptosis in lymphocytes may be inheritable . thus these results , although preliminary , need to be substantiated in larger studies . previous finding on the casp10 i522l polymorphism from a large breast cancer study showed that the variant ll genotype was associated with a borderline significant 1.30-fold increased cancer risk compared with the wild - type ii homozygote , which is consistent with the notion that the ll homozygote contributes to a diminished ac . however , the role of the extrinsic pathway ( or only casp10 ) in b[a]p - induced apoptosis was not obvious in the present study , which needs further evaluation . in conclusion , this proof - of - principle study of genotype - phenotype correlation provides evidence that potentially functional polymorphisms in the core genes of the apoptotic pathways may have a role in regulating the apoptotic response to carcinogen exposure , at least in primary lymphocytes , although there is tissue specificity in response to exposure to carcinogens . such a modification of host carcinogen - induced ac in primary lymphocytes may contribute to variation in individual susceptibility to cancer in the general population . although this study may be limited due to small sample size , multiple tests , and lack of repeated ac measurements for the same individuals , the findings , if validated in more rigorously designed and larger studies , should facilitate the design of future studies aimed at identifying subpopulations at risk of cancer and other apoptosis - related diseases .
apoptotic capacity ( ac ) in primary lymphocytes may be a marker for cancer susceptibility , and functional single nucleotide polymorphisms ( snps ) in genes involved in apoptotic pathways may modulate cellular ac in response to dna damage . to further examine the correlation between apoptotic genotypes and phenotype , we genotyped 14 published snps in 11 apoptosis - related genes ( i.e. , p53 , bcl-2 , bax , casp9 , dr4 , fas , fasl , casp8 , casp10 , casp3 , and casp7 ) and assessed the ac in response to benzo[a]pyrene-7,8 - 9,10-diol epoxide ( bpde ) in cultured primary lymphocytes from 172 cancer - free subjects . we found that among these 14 snps , r72p , intron 3 16-bp del / ins , and intron 6 g > a in p53 , 938c > a in bcl-2 , and i522l in casp10 were significant predictors of the bpde - induced lymphocytic ac in single - locus analysis . in the combined analysis of the three p53 variants , we found that the individuals with the diplotypes carrying 0 - 1 copy of the common p53 r - del - g haplotype had higher ac values compared to other genotypes . although the study size may not have the statistical power to detect the role of other snps in ac , our findings suggest that some snps in genes involved in the intrinsic apoptotic pathway may modulate lymphocytic ac in response to bpde exposure in the general population . larger studies are needed to validate these findings for further studying individual susceptibility to cancer and other apoptosis - related diseases .
You are an expert at summarizing long articles. Proceed to summarize the following text: the us health care delivery system is comprised of at least five components : cost , quality , accessibility , maintenance of the medical education system , and availability of a well trained physician workforce , all of which are being threatened by major challenges.1 among these challenges is the high cost of health care , the concern for consistent high quality medical services coupled with increased safety concerns , and lack of accessibility to primary care and specialty physicians in specific urban and rural areas . additionally , the medical education system , long based on a not - for - profit university based model , which advocated fewer schools and a rigorous curriculum , and produced fewer , but well - trained physicians , does not meet the expanding population s need for more doctors and questions the current model of the physician education and training system . currently , there is a significant physician workforce shortage in the us for which there is no quick solution.1 the size , demographics , specialty choice , and practice locations and patterns of the physician workforce are the outcome of current and past decisions made by individual physicians . their decisions were influenced by many factors including personal choice , market forces , state and federal policies and programs , and institutions which constitute the health care system and medical education infrastructure.2 thus , the nature of our physician shortage is remarkably complex . as of january 2013 , there were 5,900 designated primary care health profession shortage areas , defined by the us department of health and human services health resources and services administration as a physician to population ratio of 1:3,500.3 the physician workforce is unevenly distributed throughout the us and pockets of severe shortages occur primarily in poor , rural , and inner - city areas . the demand for an increasing number of physicians is also fueled by many factors including population growth , the aging population , changes in medical insurance coverage and type ( including the impending implementation of the affordable care act s health insurance marketplace in 2014 ) , economic growth , technological advances , and government policy . new additions to the physician workforce ( including both us and international medical graduates ( imgs ) ) and the rate of attrition ( whether through retirement , death , or disability ) determine the supply of active , practicing physicians . physicians enter practice in the us through one of three routes : graduation from a us allopathic or osteopathic school of medicine , or graduation from an international medical school . one effort within the us to address the workforce shortage has been the expansion of medical school class size and the establishment of new medical schools . us medical schools are on track to increase their enrollment thirty percent by 2017.4 concurrent to these efforts has been the increase in the number of for - profit schools , particularly in the caribbean , but now including the us . the concept of for - profit medical schools has been a source of controversy since their inception ; however , many can agree that the advantage of these schools is to augment the medical education system by adding more physician providers . the medical education system is complex as well , in that it encompasses both medical school education and residency training , each governed by its own oversight body . the association of american medical colleges ( aamc ) is the oversight organization of medical school education . the liaison committee on medical education ( lcme ) , jointly sponsored by the aamc and the council on medical education of the american medical association ( ama ) , accredits undergraduate allopathic medical schools . the accreditation council for graduate medical education ( acgme ) and the american board of medical specialties ( abms ) oversee different aspects of the graduate medical education system . the aamc represents the 141 accredited us and 17 accredited canadian medical schools . additionally , the aamc impacts roughly 400 major teaching hospitals and health systems , including 51 department of veterans affairs medical centers , and nearly 90 academic and scientific societies . the aamc represents 128,000 faculty members , 75,000 medical students , and 110,000 resident physicians.5 in regards to osteopathic medicine , the american osteopathic association ( aoa ) represents the 29 accredited colleges of osteopathic medicine and oversees the osteopathic graduate medical education ( ogme ) as well . both acgme and ogme postdoctoral training programs are recognized by the center for medicare and medicaid services , meaning that such programs receive funding from medicare and medicaid . contributing to the complexity of the overall system is the addition of imgs and for - profit medical schools ( including the caribbean medical schools ) to the pool that feeds resident physician training . the purpose of this manuscript is to define the role of for - profit medical schools in augmenting the us health care delivery system through its contributions to the medical education system and physician workforce . in order to address the potential impact of for - profit medical schools on the current and future physician workforce , it is important to review their history and evolution . the nation s first medical schools ( university of pennsylvania , harvard , and yale , to name a few ) were university - based teaching hospitals . shortly after their openings , in the early 1800s , the first for - profit medical school was established in baltimore . over the course of the next century , many more schools came into existence , both university - based and for - profit . the majority in fact , were for - profit ; only one - third of the 155 medical schools at the beginning of the 20 century were not - for - profit university - based models.6 substandard medical care punctuated this time in us history ; lax admission standards , perfunctory testing , the absence of clinical rotations , and the lack of licensing boards contributed to the poor quality of physicians produced in this era . with the founding of johns hopkins university school of medicine in 1893 , the practice of medicine was revolutionized . by applying unprecedented standards to medical training rigid entrance requirements were established and the curriculum emphasized scientific methods as well as bedside teaching and laboratory research . while other schools followed suit , many continued to adhere to the old standards of medical education , including the for - profits . during this time abraham flexner , an american educator , was commissioned by the carnegie foundation to compile a report on the medical education system . in 1910 , he presented his report titled medical education in the united states and canada : a report to the carnegie foundation for the advancement of teaching . in this report , flexner noted that too many poorly trained physicians were the end product of a multitude of commercial medical schools . such exploitation of medical education is strangely inconsistent with the social aspects of medical practice ... the medical profession is an organ differentiated by society for its highest purposes , not a business to be exploited.7 he opposed the for - profit medical school concept , instead favoring a university - based system and advocated for fewer medical schools producing quality , well - trained physicians . this standardization , which occurred in the early 1900 s , resulted in the last for - profit medical school in the continental us to close in 1930.8 it is important to attempt to define a for - profit school as there are several types . the first type is known as an educational management organization ( emo ) , which utilizes public funds in conjunction with school districts and charter schools to support primary and secondary education ( k-12 ) . the other main type of for - profit schools are those of higher education , including universities , colleges and academies , and trade and vocational schools that award academic degrees or professional certifications . simply put , for - profits are in business to be profitable for owners and shareholders by offering a service , in this case education . since the flexner era , for - profit medical schools have been met with resistance . to combine the words for - profit the obvious main concern is whether a for - profit medical school could sustain the altruistic commitment to medical education , in the face of financial challenges . thus , the term for - profit medical school remains contradictory in the eyes of many . several attempts to reinstitute a for - profit medical school in the us have been challenged and defeated , including a business venture in 1999 by ross university school of medicine in wyoming ( a for - profit allopathic medical school with roots on the caribbean island of dominica ) . to date , we have seen the establishment of one proprietary medical school in the continental us . currently , there is one for - profit medical school in the us . founded in 2006 , rocky vista university college of osteopathic medicine ( rvucom ) in parker , co is fully accredited by the american osteopathic association s commission on osteopathic college accreditation ( aoa - coca ) . their mission statement reads : rocky vista university provides quality healthcare education while inspiring students to serve with compassion , integrity , and excellence . academic admission requirements are similar to other allopathic and osteopathic medical schools , requiring a baccalaureate degree with prerequisite coursework , minimum gpa requirements , and medical college admission test ( mcat ) scores . from an educational standpoint , critics have expressed concern over the lack of academic teaching hospitals associated with the medical school.9 clinical rotations are completed at various hospitals throughout the state of colorado and the mountain west . third and fourth year medical students in their clerkships could be sent for rotations to nonacademic community hospitals without a strong background in education and research . proponents argue that rocky vista must adhere to the same educational standards as all other accredited , non - profit allopathic and osteopathic schools and will maintain the same educational outcomes.10 they state that the change lies in the structure of medical education and does not reflect a subsequent change in curriculum , clinical training , or research . from a monetary standpoint , critics claim that rvucom will be indebted to its investors , and as a result educational quality will be sacrificed.11 if standardized test scores are a reflection of educational quality , this appears to not be the case . rvucom reports a 97% comlex - i pass rate and a 98% comlex - ii pass rate , purportedly amongst the top in the nation for osteopathic schools.12 the 2012 comlex- i and comlex - ii pass rates among osteopathic schools was 92.6% and 90% , respectively.13 the founder of rocky vista , florida businessman yief tien , remains on the board of trustees as does his wife ; the scope of independence and influence of this situation remains unclear . of note , tien also served as chief operating officer of the american university of the caribbean in st . maarten until 2011 and was formerly the president and director of medical education administrative services , inc . the school s auditing firm projected no return on the initial 100 million dollar investment for several years and stated that it will take seventeen years alone to break even.9 indeed , in the 2008 fiscal year , approximately two years after the institution s inception , rvucom incurred a loss of $ 9.9 million.14 by the 2011 fiscal year , rvucom had noted a profit of $ 4.5 million.15 by these numbers , it will no doubt take some time for rvucom to break even . the 20132014 tuition rate for first year medical students is $ 46,742 ; the total cost of attendance for the first year ( tuition , health insurance , books , supplies , room and board , etc ) is estimated at $ 77,064 . for colorado residents , the total estimated cost of a four - year medical education at rocky vista is $ 305,490.16 while certainly not the most expensive of the osteopathic medical schools , it is on the high end according to 20122013 cost of attendance data from the american association of colleges of osteopathic medicine ( aacom).17 like other medical schools , rvucom participates in federal student aid programs and students finance their medical education through a combination of federal and private loans , scholarships , and personal contributions . in 2012 , rvucom s average indebtedness was $ 199,000.17 the education section of u.s . news & world report in 2010 surveyed 112 medical schools ( both allopathic and osteopathic ) , which reported an average medical school indebtedness of $ 145,020.18 the majority of schools with the heaviest debt burden of over $ 200,000 were largely private not for - profit medical schools . roughly 80% of rvucom s revenue , as with the other private osteopathic medical schools , comes from tuition and fees . in contrast , tuition and fees comprise only 14% of public osteopathic medical schools revenue.15 rvucom hopes to address the looming need for primary care physicians in the mountain west region and is based on the premise that physicians practice in locations close to their residency or fellowship programs . the first class of 159 students began studies in august 2008 . the class was evenly matched by gender and predominantly white ( 74.8% ) , a continuing trend . the latest data in total enrollment during the 20122013 academic year reports 45% female and 79.7% white students.19 with an attrition rate of approximately 21% , the inaugural class graduated 126 new physicians in may 2012 . the majority secured a residency position through the military match , the aoa match , or the acgme match . approximately 52% of the class entered primary care specialties.20 however , many of the graduates had to leave the state to complete residency training requirements , and therefore , the impact on physician workforce distribution remains unclear . table 1 lists the 2012 match results . as mentioned , the majority of for - profit medical schools exist in the caribbean . while other international for - profit schools attract us citizens , the sheer number of schools , the close proximity to the us , and the commonality of the english language draws many us medical students to the caribbean . unlike domestic schools , these schools also recruit and cater to the international populations , although the majority of students come from the us . the caribbean medical school trend began in the 1970s and was spearheaded by american entrepreneurs and medical professionals who addressed the need for medical education which could not be met domestically . this trend has continued as progressively more for - profit medical schools have opened across the caribbean over the past several decades . george s university , ross university , american university of the caribbean , and saba university schools of medicine represent a few of the more established medical schools in the region , having opened in the 1970s or 1980s . vincent and of barbados , all saints , and atlantic university schools of medicine have opened in the last two or three years alone . as of 2011 , there were sixty medical schools in the caribbean , 29 of which are regional and 31 of which are offshore . regional medical schools are designed to educate and retain graduates in the country or region where the school is located ; offshore medical schools train students from the united states who plan on returning for residency and clinical practice . the offshore medical school supply has emerged in part to meet the ever - growing demand for an increase in medical school graduates ( and ultimately an increase in the physician workforce ) . additionally , caribbean medical schools are often more appealing to those who view an allopathic doctor of medicine ( m.d . ) degree as more valuable than a stateside acquired osteopathic doctor of osteopathic ( d.o . ) degree , as all degrees awarded from these programs carry the title of doctor of medicine . while tuition rates in offshore for - profit medical schools may be high , the cost may be equivalent to or less than that of a private institution in the united states . similar to rvucom in colorado and other medical schools in the us , many caribbean medical schools participate in federal student aid programs . these schools also tend to attract those students who are unable to gain acceptance into us medical schools for various reasons , such as poor scores on standardized testing ( i.e. , the mcat ) or low undergraduate gpa s . educational standards vary , and admissions criteria are often less stringent than those required from us schools . for example , some schools such as the american university of antigua ( aua ) , the international american university ( iau ) college of medicine , and the caribbean medical university do not require the mcat . thus , acceptance rates are higher than average and class sizes are often much larger than their us counterparts . george s university , in grenada , had 554 entering students for the 2013 spring enrollment , and each incoming class at ross university in dominica accepts approximately 350 students ( there are several classes a year ) . as some of the oldest medical schools in the caribbean , st . it is likely that the attrition rate is high given each school s relatively long history ( st . other applicants choose caribbean schools simply because they are one of many highly qualified applicants vying for a limited number of medical school slots in a state with strong competition . historically , investors interested in offshore ventures were able to develop profit - generating institutions that were subject to much less stringent oversight from local authorities than in the us . a business license might be all that is required to open an offshore medical school , compared to the highly regulated financial and academic governance for a new medical school in the us . however , since the establishment of the caribbean accreditation authority in 2003 , steps have been made to oversee quality control across programs in the caribbean . nevertheless , it is still difficult to ensure standardization , as the accreditation process is voluntary . as of june 2012 , only eleven schools had subjected themselves to accreditation reviews and four have received full accreditation.22 the world education services , a non - profit organization that provides research about international education , does not consider offshore for - profit medical schools equivalent to accredited us medical schools as more time is needed to determine educational quality at these institutions.23 thirty years ago , studies demonstrated that us imgs did not perform as well on standardized tests as did graduates of us medical schools.24 a more recent study , analyzing the performance of physicians who attended caribbean medical schools , showed considerable variation in quality indicators , including ecfmg and specialty board certification rates.25 the structure of medical education in the caribbean today varies widely by school and country . united states medical licensing exam ( usmle ) scores are a good reflection of this . the usmle is a 3-part test ( step 1 , step 2 , which is divided into a clinical knowledge ( ck ) and clinical skills ( cs ) portion , and step 3 ) that assesses a physician s medical knowledge applied to clinical skills that are fundamental in providing safe and effective patient care . a physician must pass the exams before being permitted to practice medicine in the us . step 1 is usually taken after the first two years of basic science studies and just prior to starting clinical rotations . the step 2 exams are taken during the last year of medical school , and step 3 is taken following the completion of medical school . according to 2012 usmle performance data for step 1 , first time test takers from the us and canadian schools have a 96% pass rate , while the pass rate from non - us schools , including the caribbean , is 76% . similarly , the first time pass rate for step 2 ck and cs amongst american and canadian schools is 98% and 97% , respectively , while non - us schools have an 85% and 80% pass rates , respectively.26 the question remains whether the quality of medical education in the caribbean , from adequate teaching facilities equipped with up to date clinical teaching tools and research facilities to an educationally challenging curriculum taught by qualified instructors , is comparable to us medical schools.27 as direct patient care is limited on the islands , and given the sheer number of medical students , many schools have multiple affiliations with hospitals throughout the us for third and fourth year medical students in their clinical rotations . in new york for example , the number of students rotating from caribbean medical schools almost equals the number of students from the state s own medical schools.28 historically , us img match rates are much lower than those of us graduates and are illustrated in figure 1 . for us allopathic seniors , 93.7 % were matched to post graduate year ( pgy ) positions in 2013 . match rates have historically ranged from 92%94%.29 only 53.1% of us imgs matched in 2013 , however , and this marked the highest match rate since 2005 ( table 3 ) . currently , there is one for - profit medical school in the us . founded in 2006 , rocky vista university college of osteopathic medicine ( rvucom ) in parker , co is fully accredited by the american osteopathic association s commission on osteopathic college accreditation ( aoa - coca ) . their mission statement reads : rocky vista university provides quality healthcare education while inspiring students to serve with compassion , integrity , and excellence . academic admission requirements are similar to other allopathic and osteopathic medical schools , requiring a baccalaureate degree with prerequisite coursework , minimum gpa requirements , and medical college admission test ( mcat ) scores . from an educational standpoint , critics have expressed concern over the lack of academic teaching hospitals associated with the medical school.9 clinical rotations are completed at various hospitals throughout the state of colorado and the mountain west . third and fourth year medical students in their clerkships could be sent for rotations to nonacademic community hospitals without a strong background in education and research . proponents argue that rocky vista must adhere to the same educational standards as all other accredited , non - profit allopathic and osteopathic schools and will maintain the same educational outcomes.10 they state that the change lies in the structure of medical education and does not reflect a subsequent change in curriculum , clinical training , or research . from a monetary standpoint , critics claim that rvucom will be indebted to its investors , and as a result educational quality will be sacrificed.11 if standardized test scores are a reflection of educational quality , this appears to not be the case . rvucom reports a 97% comlex - i pass rate and a 98% comlex - ii pass rate , purportedly amongst the top in the nation for osteopathic schools.12 the 2012 comlex- i and comlex - ii pass rates among osteopathic schools was 92.6% and 90% , respectively.13 the founder of rocky vista , florida businessman yief tien , remains on the board of trustees as does his wife ; the scope of independence and influence of this situation remains unclear . of note , tien also served as chief operating officer of the american university of the caribbean in st . maarten until 2011 and was formerly the president and director of medical education administrative services , inc . the school s auditing firm projected no return on the initial 100 million dollar investment for several years and stated that it will take seventeen years alone to break even.9 indeed , in the 2008 fiscal year , approximately two years after the institution s inception , rvucom incurred a loss of $ 9.9 million.14 by the 2011 fiscal year , rvucom had noted a profit of $ 4.5 million.15 by these numbers , it will no doubt take some time for rvucom to break even . the 20132014 tuition rate for first year medical students is $ 46,742 ; the total cost of attendance for the first year ( tuition , health insurance , books , supplies , room and board , etc ) is estimated at $ 77,064 . for colorado residents , the total estimated cost of a four - year medical education at rocky vista is $ 305,490.16 while certainly not the most expensive of the osteopathic medical schools , it is on the high end according to 20122013 cost of attendance data from the american association of colleges of osteopathic medicine ( aacom).17 like other medical schools , rvucom participates in federal student aid programs and students finance their medical education through a combination of federal and private loans , scholarships , and personal contributions . in 2012 , rvucom s average indebtedness was $ 199,000.17 the education section of u.s . news & world report in 2010 surveyed 112 medical schools ( both allopathic and osteopathic ) , which reported an average medical school indebtedness of $ 145,020.18 the majority of schools with the heaviest debt burden of over $ 200,000 were largely private not for - profit medical schools . roughly 80% of rvucom s revenue , as with the other private osteopathic medical schools , comes from tuition and fees . in contrast , tuition and fees comprise only 14% of public osteopathic medical schools revenue.15 rvucom hopes to address the looming need for primary care physicians in the mountain west region and is based on the premise that physicians practice in locations close to their residency or fellowship programs . the class was evenly matched by gender and predominantly white ( 74.8% ) , a continuing trend . the latest data in total enrollment during the 20122013 academic year reports 45% female and 79.7% white students.19 with an attrition rate of approximately 21% , the inaugural class graduated 126 new physicians in may 2012 . the majority secured a residency position through the military match , the aoa match , or the acgme match . approximately 52% of the class entered primary care specialties.20 however , many of the graduates had to leave the state to complete residency training requirements , and therefore , the impact on physician workforce distribution remains unclear . as mentioned , the majority of for - profit medical schools exist in the caribbean . while other international for - profit schools attract us citizens , the sheer number of schools , the close proximity to the us , and the commonality of the english language draws many us medical students to the caribbean . unlike domestic schools , these schools also recruit and cater to the international populations , although the majority of students come from the us . the caribbean medical school trend began in the 1970s and was spearheaded by american entrepreneurs and medical professionals who addressed the need for medical education which could not be met domestically . this trend has continued as progressively more for - profit medical schools have opened across the caribbean over the past several decades . george s university , ross university , american university of the caribbean , and saba university schools of medicine represent a few of the more established medical schools in the region , having opened in the 1970s or 1980s . vincent and of barbados , all saints , and atlantic university schools of medicine have opened in the last two or three years alone . as of 2011 , there were sixty medical schools in the caribbean , 29 of which are regional and 31 of which are offshore . regional medical schools are designed to educate and retain graduates in the country or region where the school is located ; offshore medical schools train students from the united states who plan on returning for residency and clinical practice . the offshore medical school supply has emerged in part to meet the ever - growing demand for an increase in medical school graduates ( and ultimately an increase in the physician workforce ) . additionally , caribbean medical schools are often more appealing to those who view an allopathic doctor of medicine ( m.d . ) degree as more valuable than a stateside acquired osteopathic doctor of osteopathic ( d.o . ) degree , as all degrees awarded from these programs carry the title of doctor of medicine . while tuition rates in offshore for - profit medical schools may be high , the cost may be equivalent to or less than that of a private institution in the united states . similar to rvucom in colorado and other medical schools in the us , many caribbean medical schools participate in federal student aid programs . these schools also tend to attract those students who are unable to gain acceptance into us medical schools for various reasons , such as poor scores on standardized testing ( i.e. , the mcat ) or low undergraduate gpa s . educational standards vary , and admissions criteria are often less stringent than those required from us schools . for example , some schools such as the american university of antigua ( aua ) , the international american university ( iau ) college of medicine , and the caribbean medical university do not require the mcat . thus , acceptance rates are higher than average and class sizes are often much larger than their us counterparts . george s university , in grenada , had 554 entering students for the 2013 spring enrollment , and each incoming class at ross university in dominica accepts approximately 350 students ( there are several classes a year ) . as some of the oldest medical schools in the caribbean , st . george s and ross it is likely that the attrition rate is high given each school s relatively long history ( st . other applicants choose caribbean schools simply because they are one of many highly qualified applicants vying for a limited number of medical school slots in a state with strong competition . historically , investors interested in offshore ventures were able to develop profit - generating institutions that were subject to much less stringent oversight from local authorities than in the us . a business license might be all that is required to open an offshore medical school , compared to the highly regulated financial and academic governance for a new medical school in the us . however , since the establishment of the caribbean accreditation authority in 2003 , steps have been made to oversee quality control across programs in the caribbean . nevertheless , it is still difficult to ensure standardization , as the accreditation process is voluntary . as of june 2012 , only eleven schools had subjected themselves to accreditation reviews and four have received full accreditation.22 the world education services , a non - profit organization that provides research about international education , does not consider offshore for - profit medical schools equivalent to accredited us medical schools as more time is needed to determine educational quality at these institutions.23 thirty years ago , studies demonstrated that us imgs did not perform as well on standardized tests as did graduates of us medical schools.24 a more recent study , analyzing the performance of physicians who attended caribbean medical schools , showed considerable variation in quality indicators , including ecfmg and specialty board certification rates.25 the structure of medical education in the caribbean today varies widely by school and country . united states medical licensing exam ( usmle ) scores are a good reflection of this . the usmle is a 3-part test ( step 1 , step 2 , which is divided into a clinical knowledge ( ck ) and clinical skills ( cs ) portion , and step 3 ) that assesses a physician s medical knowledge applied to clinical skills that are fundamental in providing safe and effective patient care . a physician must pass the exams before being permitted to practice medicine in the us . step 1 is usually taken after the first two years of basic science studies and just prior to starting clinical rotations . the step 2 exams are taken during the last year of medical school , and step 3 is taken following the completion of medical school . according to 2012 usmle performance data for step 1 , first time test takers from the us and canadian schools have a 96% pass rate , while the pass rate from non - us schools , including the caribbean , is 76% . similarly , the first time pass rate for step 2 ck and cs amongst american and canadian schools is 98% and 97% , respectively , while non - us schools have an 85% and 80% pass rates , respectively.26 the question remains whether the quality of medical education in the caribbean , from adequate teaching facilities equipped with up to date clinical teaching tools and research facilities to an educationally challenging curriculum taught by qualified instructors , is comparable to us medical schools.27 as direct patient care is limited on the islands , and given the sheer number of medical students , many schools have multiple affiliations with hospitals throughout the us for third and fourth year medical students in their clinical rotations . in new york for example , the number of students rotating from caribbean medical schools almost equals the number of students from the state s own medical schools.28 historically , us img match rates are much lower than those of us graduates and are illustrated in figure 1 . for us allopathic seniors , 93.7 % were matched to post graduate year ( pgy ) positions in 2013 . match rates have historically ranged from 92%94%.29 only 53.1% of us imgs matched in 2013 , however , and this marked the highest match rate since 2005 ( table 3 ) . currently , there is a significant physician workforce shortage in the us for which there is no easy solution.1 the impact of for - profit medical schools on physician workforce could play a role in minimizing this shortage , however the limiting factor remains that graduates of offshore caribbean schools often face great difficulty in securing a residency position through the us - based national resident matching program ( nrmp ) . it is generally accepted that many residency directors in the us prefer us medical graduates to imgs . the end result is that these students may go unmatched , or settle for a residency position that does not fulfill their career goals . candidates must have an educational commission for foreign medical graduates ( ecfmg ) certification , which requires completion of usmle step 1 and both portions of usmle step 2 as well as a medical degree from an institution that is registered with the international medical education directory ( imed ) . the ecfmg certification ensures that imgs are prepared to enter acgme training programs in the us . us medical students need not have their medical diplomas to enter the match during their final year of medical school . thus , stateside trained graduates can begin residency right after graduation , whereas imgs must wait several months before starting training due to the cyclical nature of the match process . the nrmp recently released data from the 2013 main residency match.29 of 26,392 pgy-1 positions available , 25,463 positions were filled . us us imgs , including the for - profit medical schools in the caribbean , filled 2,706 ( 10.6% ) of the positions . non - us imgs filled 3,601 ( 14% ) of all available positions.29 in the recent match , 1,750 ( 64.7% ) of us img applicants were accepted into primary care specialties ( e.g. , internal medicine , family medicine , and pediatrics ) . of the 2,914 family medicine positions that were filled in the match , 690 were filled by us imgs , representing roughly 24% of family medicine positions . of the 6,242 categorical internal medicine positions that were filled , 868 were filled by us imgs , representing roughly 14% of internal medicine positions . of the 2,606 pediatric positions that were filled , because of the overall physician workforce shortage , it has been suggested that a partial solution could be recruitment of imgs from other countries ( defined as non - us imgs ) . in addition , physician assistants and nurse practitioners are working to fill the gap and have helped to compensate for the physician workforce shortfall . the medical education system educates medical school graduates in primary care or specialty areas that eventually feed the physician workforce . while there is a push to expand existing medical schools or start new medical schools , the reality is that there is no effort by the federal government to increase the number of residency positions . it is important to note that the vast majority of funding for resident training comes from the department of health and human services , primarily through medicare . there has been a moratorium on all new resident positions funded through the federal government since the balanced budget act of 1997 . thus , the number of government funded residency positions has been capped for 16 years at this point . historically , hospitals funded the training of residents before medicare subsidies existed and some hospitals now fund some residency training slots . this accounts , however , for only a marginal increase in residency positions . as a result , there will not be enough positions for all medical school graduates and the need to increase the physician workforce will not occur at a rate to meet the demand . the annual medical school enrollment survey , conducted by the aamc s center for workforce studies , found that 40% of medical school deans were extremely concerned about enrollment growth outpacing the number of available residency training positions.4 in the 2013 match , there were more unmatched us seniors than unfilled positions . this disturbing statistic has happened only once before , in 2010.4 aamc president and ceo dr . darrell g. kirch s statement on this issue reflects the need for urgency in the call to increase residency positions : we re pleased to see our nation s medical schools increasing enrollment to address the projected physician shortage , but as we saw in the results of this year s match , congress now needs to do its part and act quickly to increase the number of federally funded residency training positions in order for all medical school graduates to be able to complete their training and become practicing physicians.4 thus , unless the federal government works to lift the cap on residency training positions , the plethora of medical school graduates , from for - profits or otherwise , will not be able to impact on the growing gap between the physician workforce and the public need . the availability of a well - trained physician workforce and maintaining the medical education system are just two of several components of the health care delivery system that are intricately linked . in order to effect change in one , for - profit medical schools represent a novel , albeit controversial , approach to solving this dilemma . the impact of the only for - profit medical school in the us remains to be seen . offshore for - profit medical schools , including those in the caribbean , continue to provide a large number of medical school graduates who return to the us for postgraduate medical training . however , these students are also subject to the same residency caps that have and will continue to affect the number of available residency training positions in the us . in the meantime , we will continue to face the challenges of supply vs. demand in providing a well - trained , adequate physician workforce to a us population that should have ready access to quality healthcare .
currently , in the united states there is a significant physician workforce shortage . this problem is likely to persist as there is no quick solution . the nature of this shortage is complex and involves factors such as an absolute physician shortage , as well as physician shortages in primary care and certain specialty care areas . in addition , there is a misdistribution of physicians to medically underserved areas and populations . the medical education system trains medical school graduates that eventually feed the physician workforce . however , several factors are in place which ultimately limits the effectiveness of this system in providing an appropriate workforce to meet the population demands . for - profit medical schools have been in existence in and around the continental us for many years and some authors have suggested that they may be a major contributor to the physician workforce shortage . there is currently one for - profit medical school in the us , however the majority exist in the caribbean . the enrollment in and number of these schools have grown to partially meet the ever - growing demand for an increase in medical school graduates and they continue to provide a large number of graduates who return to the us for postgraduate medical training and , ultimately , increase the physician workforce . the question is whether this source will benefit the workforce quality and quantity needs of our growing and aging population .
You are an expert at summarizing long articles. Proceed to summarize the following text: anorexia nervosa ( anorexia ) is an eating disorder characterized by a distorted perception of the body shape and an intense fear of gaining weight , which results in abnormally low body weight and various medical complications ( 1,2 ) . previous reports have shown that as many as 70% of anorexia patients had associated renal problems , including a decreased glomerular filtration rate , impaired concentrating ability , and electrolyte abnormalities ( 3 - 5 ) . the pathophysiology of chronic kidney disease ( ckd ) in anorexia patients is not yet fully understood , although several previous renal biopsy reports revealed chronic tubulointerstitial nephritis ( tin ) that is probably due to prolonged hypokalemia ( 4 - 6 ) . we herein present the renal biopsy findings of an anorexia patient who showed acute exacerbation of ckd . the patient was a 31-year - old woman who had been suffering from an eating disorder since she was 13 years old . she had been vomiting repeatedly on purpose and abused diuretics and alcohol . at age 26 , hypokalemia ( serum potassium of 2.8 mmol / l ) were noted , and she was introduced to our outpatient clinic . at the first visit , she showed chronic kidney disease ( serum creatinine of 1.18 mg / dl ) and hyperuricemia ( serum uric acid of 11.7 mg / dl ) . she had been prescribed potassium chloride tablets and an antihyperuricemic agent ( febuxostat ) at the outpatient clinic , but she did not come to the hospital regularly . she did not take any prescribed medications regularly and probably took non - prescribed medications such as diuretics in secret . she sometimes had acute kidney injury due to dehydration , which soon resolved after fluid replacement , and her serum creatinine level had been stable around 1.5 - 2.0 mg / dl . however , at age 31 , she complained of a gout attack and showed acute kidney injury ( serum creatinine of 8.95 mg / dl ) . ultrasound revealed that both of her kidneys were slightly atrophic ( major axis was around 9.5 cm ) , and no hydronephrosis was found . we gave her fluid replacement therapy , and her kidney function recovered , and her serum creatinine level dropped to 7 mg / dl , but it did not recover further . as the exacerbation of the renal function was acute , there was a possibility of acute drug - induced tin , which can respond to steroid therapy . the patient denied any non - prescription drug use and hoped to discover the reason for the acute kidney injury . therefore , we conducted renal biopsy after explaining the risks . on the day of biopsy , her height was 157 cm , weight was 44.6 kg , and body mass index ( bmi ) was 18.1 kg / m . her body temperature was 36.5 , blood pressure was 112/52 mmhg , pulse rate was 78 beats per minute , and peripheral oxygen saturation was 97% ( room air ) . laboratory tests ( table ) showed blood urea nitrogen ( bun ) of 39.3 mg / dl , serum creatinine of 8.74 mg / dl , serum potassium of 3.0 mmol / l , serum chloride of 77 mmol / l and serum bicarbonate of 55.5 mmol / l . anti - nuclear antibody ( ana ) , anti - neutrophil cytoplasmic antibody ( anca ) and anti - glomerular basement membrane ( gbm ) antibody were negative , and the serum complement ( c3 , c4 and ch50 ) levels were not decreased . the urine protein was 1.35 g / day ( selectivity index was 0.25 ) , and urine 2-microglobulin was 61,952 the urine sediment contained 5 - 9 red blood cells / high power field and some crystal casts with no uric acid salt . wbc : white blood cell , neutro : neutrophil , lymph : lymphocyte , mono : monocyte , eosino : eosinophil , baso : basophil , rbc : red blood cell , hb : hemoglobin , hct : hematocrit , mcv : mean corpuscular volume , plt : platelet , tp : total protein , alb : albumin , t - chol : total - cholesterol , bun : blood urea nitrogen , cre : creatinine , egfr : estimated glomerular filtration rate , ua : uric acid , na : sodium , k : potassium , cl : chloride , ca : calcium , ip : inorganic phosphorus , fbs : fasting blood sugar , ph : power of hydrogen , paco2 : partial pressure of arterial carbon dioxide , hco3 : bicarbonate , be : base excess , crp : c - reactive protein , ana : anti - nuclear antibody , igg : immunoglobulin g , iga : immunoglobulin a , igm : immunoglobulin m , aso : anti - streptolysin o , anca : anti - neutrophil cytoplasmic antibody , anti - gbm : anti - glomerular basement membrane antibody , bjp : bence jones protein , 2 mg : 2-microglobulin the renal biopsy specimens included 41 glomeruli . fifteen glomeruli had normal features , 23 showed complete sclerosis , and 2 showed segmental sclerosis ( fig . on immunofluorescent staining , the glomeruli were negative for igg , iga , igm , c1q , c3 , c4 , and fibrinogen . on electron microscopy , collapsed glomerular capillaries and wrinkled glomerular basement membranes were noted . ( a ) chronic tubulointerstitial nephritis ( tin ) and collapse of glomeruli ; periodic acid - schiff stain ( 100 ) . ( b ) secondary perihilar glomerulosclerosis ; periodic acid - schiff stain ( 400 ) . ( c , ( e ) widespread infiltration of many lymphocytes and plasma cells into the interstitium ; hematoxylin and eosin staining ( 400 ) . ( f ) the tubular basement membranes ( tbms ) were thickened , and the epithelium was flattened with complete loss of the brush border ( obsolete tin , upper half ) . lymphocytes infiltrated into the tubules , but the structures of the tubules were intact ( active tin , lower half ) ; periodic acid - schiff stain ( 400 ) . numerous lymphocytes and plasma cells the tubular structures were intact , and there was scattered mild lymphocytic infiltration of the tubules ( fig . in some areas , a collection of atrophic tubules with thickened basement membranes and flattened epithelia with complete loss of the brush border were found ( fig . 1f , upper half ) . the crystals in the distal tubules had a high density ( fig . 2c and d ) . ( a ) a number of crystals broadly deposited in the distal tubules were stained basophilic by hematoxylin and eosin staining ( 400 ) . ( b ) the crystals were stained black by von kossa stain ( 400 ) . ( c , d ) the crystals in the distal tubules had a high density on electron microscopy ( 1,500 , 1,200 ) . these histologic findings were considered indicative of protracted active tin , secondary glomerulosclerosis , and tubular calcium depositions . renal biopsy showed tin with calcification of the distal tubules but did not reveal the exact reason for the acute exacerbation of ckd . given the observation of active tin with interstitial expansion and tubulitis , we recommended that she undergo steroid therapy , but she declined . we continued conservative treatment , and her renal function temporarily improved , as her serum creatinine dropped to 4 mg / dl in 6 months . since then , her renal function has fluctuated , occasionally approaching end - stage kidney disease . fifteen glomeruli had normal features , 23 showed complete sclerosis , and 2 showed segmental sclerosis ( fig . on immunofluorescent staining , the glomeruli were negative for igg , iga , igm , c1q , c3 , c4 , and fibrinogen . on electron microscopy , collapsed glomerular capillaries and wrinkled glomerular basement membranes were noted . ( a ) chronic tubulointerstitial nephritis ( tin ) and collapse of glomeruli ; periodic acid - schiff stain ( 100 ) . ( b ) secondary perihilar glomerulosclerosis ; periodic acid - schiff stain ( 400 ) . ( c , ( e ) widespread infiltration of many lymphocytes and plasma cells into the interstitium ; hematoxylin and eosin staining ( 400 ) . ( f ) the tubular basement membranes ( tbms ) were thickened , and the epithelium was flattened with complete loss of the brush border ( obsolete tin , upper half ) . lymphocytes infiltrated into the tubules , but the structures of the tubules were intact ( active tin , lower half ) ; periodic acid - schiff stain ( 400 ) . numerous lymphocytes and plasma cells the tubular structures were intact , and there was scattered mild lymphocytic infiltration of the tubules ( fig . 1f , lower side ) . in some areas , a collection of atrophic tubules with thickened basement membranes and flattened epithelia with complete loss of the brush border were found ( fig . 1f , upper half ) . the crystals in the distal tubules had a high density ( fig . 2c and d ) . ( a ) a number of crystals broadly deposited in the distal tubules were stained basophilic by hematoxylin and eosin staining ( 400 ) . ( b ) the crystals were stained black by von kossa stain ( 400 ) . ( c , d ) the crystals in the distal tubules had a high density on electron microscopy ( 1,500 , 1,200 ) . these histologic findings were considered indicative of protracted active tin , secondary glomerulosclerosis , and tubular calcium depositions . renal biopsy showed tin with calcification of the distal tubules but did not reveal the exact reason for the acute exacerbation of ckd . given the observation of active tin with interstitial expansion and tubulitis , we recommended that she undergo steroid therapy , but she declined . we continued conservative treatment , and her renal function temporarily improved , as her serum creatinine dropped to 4 mg / dl in 6 months . since then , her renal function has fluctuated , occasionally approaching end - stage kidney disease . the renal biopsy findings of this patient provide two important clinical suggestions : chronic tin caused by anorexia does not uniformly progress , and not only tin but also widespread calcification of the distal tubules can aggravate the renal function of anorexia patients . renal biopsy of this patient revealed that some areas were obsolete lesions and other areas were active lesions . it was previously reported that anorexia was associated with chronic tin ( 5 - 7 ) . the main cause of chronic tin is considered to be prolonged hypokalemia , which may be caused by a deficit of potassium in the diet , diuretic abuse , repeated vomiting , and stimulation of the renin - angiotensin - aldosterone system ( 8) . prolonged hypokalemia is known to produce vacuolar lesions in the epithelial cells of tubules ( 9 ) , ultimately leading to tin ( 4,10,11 ) , the mechanism of which has been partially clarified by animal experiments ( 12 - 15 ) . since the present patient showed serum potassium of 2.5 - 3.5 mmol / l , prolonged hypokalemia might have an important role in the progression of tin . however , considering that the chronic tin of this patient did not uniformly progress , prolonged hypokalemia might not be the only factor that induced tin . it was previously reported that not only prolonged hypokalemia but also chronic reduced kidney perfusion , repeated urinary tract infection , and drug abuse were related to tin in anorexia patients ( 8) . this patient 's tin might have been induced by many factors related to anorexia . the renal function might recover after steroid use because of the existence of active tin , but steroid therapy has not been used to treat chronic tin of anorexia patients in previous reports . second , not only tin but also widespread calcification of the distal tubules might have aggravated the renal function of anorexia patients . renal biopsy of this patient showed that a number of crystals , including calcium ones , were widely deposited in the distal tubules . since the serum calcium level and the amount of urine calcium ( 0.06 g / day ) were within the normal range , the likelihood of diseases that cause hypercalciuria , such as hyperparathyroidism and sarcoidosis , was low . previous reports have suggested that anorexia patients tend to show nephrocalcinosis ( 16 - 18 ) . furosemide inhibits na - k-2cl cotransporters in the thick ascending limb of the loop of henle , increases the intraluminal calcium concentration , and causes nephrocalcinosis ( 19 ) , although we did not determine the exact amount of diuretics this patient had been taking or for how long . an autopsy report of an anorexia patient suggested that an enhanced renin - angiotensin system inhibited napi - iia cotransporters in the proximal tubules and ca - atpase in the thin limb of the loop of henle , causing nephrocalcinosis ( 17,18 ) . these previous reports did not detect an association between nephrocalcinosis and aggravation of the renal function . however , renal biopsy of this patient showed that the deposition of many crystals in the distal tubules was very widespread , which might have contributed to the aggravation of the renal function by itself . in conclusion , chronic tin caused by anorexia does not uniformly progress , and not only tin but also widespread calcification of the distal tubules might aggravate the renal function of anorexia patients . an accumulation of further cases is needed to understand the progression of ckd caused by anorexia .
although patients with anorexia nervosa ( anorexia ) are known to show tubulointerstitial nephritis ( tin ) , the pathophysiology of its progression is not fully understood . we herein report a 31-year - old woman with anorexia who showed acute exacerbation of chronic kidney disease . renal biopsy showed non - uniform chronic tin ; some areas were obsolete lesions and other areas were active lesions . in addition , many calcium - containing crystals were widely deposited in the distal tubules . the results suggest that chronic tin in the setting of anorexia does not uniformly progress and that not only tin but also widespread calcification of distal tubules might aggravate the renal function of anorexia patients .
You are an expert at summarizing long articles. Proceed to summarize the following text: diarrhea is the fourth - leading cause of death among children under 5 y of age worldwide , killing an estimated 530,000 and hospitalizing millions in 2015 , mostly in developing countries . among the various causes of diarrheal diseases , etec and shigella recent impact studies indicate that immunizing against these two causes of diarrhea could benefit global public health significantly , particularly if they could be combined into a single vaccine for efficient immunization . such a vaccine would improve the health of infants and children in developing countries , and also could protect travelers and military personnel in areas where these bacteria are endemic . the oral route for administration of vaccines to infants is simpler , safer , and more acceptable than needle and syringe , and is particularly useful in developing countries that lack highly trained health care workers and face difficulty in disposing of sharps waste . the target tissue for oral vaccines is the mucosa of the small intestine ; however , the low ph of gastric fluid can degrade protein antigens and some adjuvant components of vaccine formulations before they reach the gut . the ability of antigens to resist degradation during transit through the stomach environment can have a profound effect on their ability to induce protective antibodies . because infants as young as 24 weeks gestational age are able to maintain the intragastric ph at the adult level below 4from the first day of life , even vaccines for young infants must survive this environment . thus , oral vaccines typically require a buffer , which can be given with the vaccine or incorporated into the formulation . oral enteric vaccine candidates in development include whole - cell inactivated bacterial and subunit vaccines most of which require a mucosal adjuvant to improve efficacy . although neutralizing stomach acid may not be as important for inactivated vaccines as for live ones , the stability of mucosal adjuvants in stomach acid must be evaluated , as some of these are sensitive to ph . the choice of buffer is important not only for maintaining vaccine efficacy , but also for avoiding unwanted side effects in infants . sodium bicarbonate , a commonly used antacid buffer , has been reported to cause discomfort from release of carbon dioxide gas after neutralization of stomach acid . this gas build - up in the stomach can create pressure that results in reflux of stomach contents into the esophagus , causing spitting , and may require re - administration of the vaccine . children can have reflux at any age , although it is more common in infants less than one year of age . other antacid buffers , such as bicarbonate - ascorbic acid , have been evaluated in clinical studies in infants and have been reported to cause less bloating than bicarbonate buffer alone . the phosphate - citrate buffer used in rotateq vaccine is another good candidate for the new enteric vaccines for infants . this buffer has sufficient stomach acid neutralizing capacity in a 2-ml dose volume , without unwanted side effects , and has shown good compatibility with different types of delivery containers . figure 1image of an infant receiving oral rotavirus vaccine from a prefilled polymer squeeze tube . image of an infant receiving oral rotavirus vaccine from a prefilled polymer squeeze tube . keeping the dose volume of vaccine formulation and buffer low is important for infants , since swallowing is slow and uncoordinated , and this can lead to spitting or vomiting of the vaccine . the total volume of the vaccine / adjuvant formulation and the buffer should not exceed 2 ml . the trivalent oral polio vaccine ( opv ) that requires 2 drops per dose is a gold standard formulation in terms of keeping the volume minimal ; however , opv is resistant to gastric acid and does not require an antacid buffer , allowing for this small dose volume . this will probably not be the case for new enteric vaccine candidates , such as a possible combination vaccine against etec and shigella , where the final vaccine presentation with multiple antigens , a mucosal adjuvant , and for vaccines formulated with antacid buffer , the largest contribution to overall dosing volume comes from the buffer component . the final formulation , comprising the vaccine and the adjuvant components , requires physiological ph and isotonicity ; thus , the buffer component must have low salt concentration to maintain viability of the antigens . at isotonic salt concentration , there is insufficient or low buffering capacity , which means large dose volumes of the formulation would be needed to effectively buffer the stomach acid . one way to maintain the low dose volume requirement for new combination vaccines is by separating the vaccine components . for example , antigens and adjuvant can be combined in one primary container , and the buffer concentrated in a low dose volume can have its own primary container . multiple administration steps are needed to fully vaccinate the infant , introducing greater risk of incomplete or improper administration of the full dose . requirements for containers and delivery devices for new oral vaccines include protection of and compatibility with the vaccine formulation , performance and ease of use in dose administration , safety , manufacturability , immunization program suitability , and cost . the stability of any new vaccine must be established for the specific material , design , and filling process for the intended container . glass vials are a common primary container for many injectable vaccines , and are also used for opv and some oral rotavirus vaccines . vials for oral administration are generally packaged with droppers to be attached at the point of use ; this approach is most suitable for very low - volume doses delivered by counting drops . polymer containers also are widely used for pharmaceuticals and for vaccines ; these include preformed containers that are sterilized and shipped to vaccine manufacturers for filling and sealing and blow - fill - seal containers that are formed , filled , and sealed in a single operation . polymer squeeze tubes are used as primary packaging for several opv and rotavirus vaccines and have the advantage of acting as the vaccine administration device , eliminating the need for an additional component for delivery . however , some adjuvants may adsorb to polymers , which would require modifying excipients to minimize this risk . polymer materials also are more permeable than glass to water vapor and oxygen transmission , and to leaching of label materials . protective secondary packaging , such as a foil pouch , may be used to prevent water and gas transfer through the container , and labeling can be applied to a tab rather than to the body of the container to minimize leaching of ink and adhesive components into the drug substance although both of these will increase the container 's cold chain volume . inclusion of a vaccine vial monitor ( vvm ) in the vaccine 's labeling is a requirement for vaccines assessed for world health organization ( who ) prequalification , in accordance with the programmatic suitability of vaccine candidates for who prequalification ( pspq ) , which provides guidelines on vaccine characteristics that will be considered during the prequalification process . for presentations requiring protective secondary packaging , placement of the vvm if multiple doses of vaccine are packaged together in the same pouch or overwrap , the vvm should be placed on the primary container so that it remains with the vaccine until each dose is delivered . however , in this case the vvm may be obscured by the secondary packaging , which could present challenges for inspection during storage . many of the features discussed here also provide safety and program suitability in low - resource settings , in addition to ease of use . for all new oral vaccine presentations , human factors and usability evaluations should be performed to assess ease of use , correct dosing , and any potential risks or errors resulting from the presentation . one important ergonomic factor for delivery of oral presentations is squeezability of the delivery device , which is determined by container shape , material stiffness , and viscosity of the vaccine . optimal usability and safety are achieved by minimizing the steps ( and subsequent risks ) associated with preparing and delivering the vaccine at the point of use , and by giving the vaccinator fine - tuned control over the dispensing speed . presentations requiring reconstitution or mixing of vaccine components at the point of use increase the time required for and the complexity of the delivery process , and have resulted in errors such as use of incorrect diluents or administration of the diluent alone by practitioners who mistake it for the complete vaccine . to this end , a critical characteristic of the pspq is that oral vaccine formulations be in a ready - to - use format ( no reconstitution needed ) for developing - country , public - sector immunization programs . compliance with this requirement is compulsory , but requests for exceptions are reviewed by the pspq standing committee , taking into account the public health need . if preparation steps are necessary , the mixing process and containers should be easy to use and should minimize the risk of errors , and all required components should be disposable and packaged with each dose of vaccine . this co - packaging requirement applies to water for reconstitution as well , as clean water may not be available at immunization sites in low - resource settings . integrated reconstitution technologies in which multiple liquid or dry vaccine components are packaged within the same primary container and mixed within the device prior to administration can simplify the preparation process and reduce the risk of errors , but can also be costlier . some features that impart ease of use to containers and delivery devices such as ready - to - use formulations also increase safety of use . an inherent safety feature of oral vaccines is that they are administered without needles , and thus needlestick injuries and sharps disposal can be avoided . another safety consideration is the use of packaging and delivery - device designs that reduce the risk of accidental injection of an oral vaccine , which can cause adverse events and render vaccination ineffective . this error has occurred with oral rotavirus vaccines , particularly those in packaging that appear similar to vaccines for injection . the risk of injection precludes use of oral vaccine presentations in glass vials intended for delivery with a syringe with the needle removed . manufacturing feasibility and cost are also important for selecting containers and delivery devices for oral vaccines . for vaccines that may be marketed to adult travelers as well as provided to infants in low - resource settings , different presentations and different final manufacturing processes may be needed . polymer containers may have lower per - unit costs than glass vials and be easier to use , but since manufacturers are likely to have glass - vial - filling facilities in place , using an alternative fill - finish process will entail start - up costs , could require building or repurposing facilities , and might increase a company 's technical burden and risks . for vaccines needing reconstitution , integrated reconstitution devices can simplify delivery but may increase manufacturing complexity and device costs . if a manufacturer is contracting with other companies for packaging or delivery components , having only a single source supplier is a risk , since a problem at this source could hold up the entire production process . immunization program suitability . for example , accurate dose preparation and measurement , as well as simple , controlled vaccine administration are desirable not only for ease of use but for program suitability in low - resource settings where health care workers may have minimal training . use of a polymer squeeze tube as the primary container and delivery device increases safety , addresses ease of use , and reduces packaging volume all of which contribute to program suitability . single - dose presentations offer advantages in ease of use , as they do not require measurement of doses ; however , they can increase manufacturing costs and cold chain storage volumes . multi - dose presentations reduce space needed in the cold chain , but they increase vaccine wastage , particularly if the vaccine is at risk of contamination or is not stable for more than one immunization session after opening . tradeoffs such as these must be assessed in determining the optimal primary container for a new vaccine . vaccine manufacturers as well as developers of packaging and administration components can get feedback on the suitability of proposed presentations of vaccines in development from the immunization practices advisory committee 's delivery technologies working group and from the who prequalification group . immunization program managers should ideally assess the overall program cost to deliver a vaccine in different presentations , in addition to vaccine price itself . some technologies may increase safety or ease of use , but likely will increase vaccine purchase costs and the burden on the cold chain , so these attributes must be balanced in selection of a vaccine presentation . developers will want to design primary containers and delivery devices with the needs of manufacturers and immunization programs in mind , in order to maximize suitability while minimizing costs . for example , for polymer tubes , a multi - mono - dose configuration in which multiple , conjoined , single - dose containers share a single label and vvm can reduce manufacturing costs and demands on storage space . however , this approach requires a design that prevents users from removing a single container without the label information . in summary , oral vaccines against bacterial pathogens such as etec and shigella that cause diarrhea in millions of children in low - resource countries could save lives and reduce the burden of serious illnesses . new oral enteric vaccine candidates must be buffered against stomach acid , formulated in minimal dose volumes , filled into containers that protect antigens and adjuvants but minimize the load on the cold chain , and can be safely administered in the correct dose by minimally trained vaccinators working in challenging environments all within the constraints of manufacturability and attention to cost . product designers who take these features into account in their development processes will be able to position new oral vaccines for implementation in routine immunization programs , and these programs in turn will achieve optimal vaccine impact . the oral route for administration of vaccines to infants is simpler , safer , and more acceptable than needle and syringe , and is particularly useful in developing countries that lack highly trained health care workers and face difficulty in disposing of sharps waste . the target tissue for oral vaccines is the mucosa of the small intestine ; however , the low ph of gastric fluid can degrade protein antigens and some adjuvant components of vaccine formulations before they reach the gut . the ability of antigens to resist degradation during transit through the stomach environment can have a profound effect on their ability to induce protective antibodies . because infants as young as 24 weeks gestational age are able to maintain the intragastric ph at the adult level below 4from the first day of life , even vaccines for young infants must survive this environment . thus , oral vaccines typically require a buffer , which can be given with the vaccine or incorporated into the formulation . oral enteric vaccine candidates in development include whole - cell inactivated bacterial and subunit vaccines most of which require a mucosal adjuvant to improve efficacy . although neutralizing stomach acid may not be as important for inactivated vaccines as for live ones , the stability of mucosal adjuvants in stomach acid must be evaluated , as some of these are sensitive to ph . the choice of buffer is important not only for maintaining vaccine efficacy , but also for avoiding unwanted side effects in infants . sodium bicarbonate , a commonly used antacid buffer , has been reported to cause discomfort from release of carbon dioxide gas after neutralization of stomach acid . this gas build - up in the stomach can create pressure that results in reflux of stomach contents into the esophagus , causing spitting , and may require re - administration of the vaccine . children can have reflux at any age , although it is more common in infants less than one year of age . other antacid buffers , such as bicarbonate - ascorbic acid , have been evaluated in clinical studies in infants and have been reported to cause less bloating than bicarbonate buffer alone . the phosphate - citrate buffer used in rotateq vaccine is another good candidate for the new enteric vaccines for infants . this buffer has sufficient stomach acid neutralizing capacity in a 2-ml dose volume , without unwanted side effects , and has shown good compatibility with different types of delivery containers . figure 1image of an infant receiving oral rotavirus vaccine from a prefilled polymer squeeze tube . keeping the dose volume of vaccine formulation and buffer low is important for infants , since swallowing is slow and uncoordinated , and this can lead to spitting or vomiting of the vaccine . the total volume of the vaccine / adjuvant formulation and the buffer should not exceed 2 ml . the trivalent oral polio vaccine ( opv ) that requires 2 drops per dose is a gold standard formulation in terms of keeping the volume minimal ; however , opv is resistant to gastric acid and does not require an antacid buffer , allowing for this small dose volume . this will probably not be the case for new enteric vaccine candidates , such as a possible combination vaccine against etec and shigella , where the final vaccine presentation with multiple antigens , a mucosal adjuvant , and a diluent / buffer may result in a large oral dose volume . for vaccines formulated with antacid buffer , the final formulation , comprising the vaccine and the adjuvant components , requires physiological ph and isotonicity ; thus , the buffer component must have low salt concentration to maintain viability of the antigens . at isotonic salt concentration , there is insufficient or low buffering capacity , which means large dose volumes of the formulation would be needed to effectively buffer the stomach acid . one way to maintain the low dose volume requirement for new combination vaccines is by separating the vaccine components . for example , antigens and adjuvant can be combined in one primary container , and the buffer concentrated in a low dose volume can have its own primary container . multiple administration steps are needed to fully vaccinate the infant , introducing greater risk of incomplete or improper administration of the full dose . requirements for containers and delivery devices for new oral vaccines include protection of and compatibility with the vaccine formulation , performance and ease of use in dose administration , safety , manufacturability , immunization program suitability , and cost . the stability of any new vaccine must be established for the specific material , design , and filling process for the intended container . glass vials are a common primary container for many injectable vaccines , and are also used for opv and some oral rotavirus vaccines . vials for oral administration are generally packaged with droppers to be attached at the point of use ; this approach is most suitable for very low - volume doses delivered by counting drops . polymer containers also are widely used for pharmaceuticals and for vaccines ; these include preformed containers that are sterilized and shipped to vaccine manufacturers for filling and sealing and blow - fill - seal containers that are formed , filled , and sealed in a single operation . polymer squeeze tubes are used as primary packaging for several opv and rotavirus vaccines and have the advantage of acting as the vaccine administration device , eliminating the need for an additional component for delivery . however , some adjuvants may adsorb to polymers , which would require modifying excipients to minimize this risk . polymer materials also are more permeable than glass to water vapor and oxygen transmission , and to leaching of label materials . protective secondary packaging , such as a foil pouch , may be used to prevent water and gas transfer through the container , and labeling can be applied to a tab rather than to the body of the container to minimize leaching of ink and adhesive components into the drug substance although both of these will increase the container 's cold chain volume . inclusion of a vaccine vial monitor ( vvm ) in the vaccine 's labeling is a requirement for vaccines assessed for world health organization ( who ) prequalification , in accordance with the programmatic suitability of vaccine candidates for who prequalification ( pspq ) , which provides guidelines on vaccine characteristics that will be considered during the prequalification process . for presentations requiring protective secondary packaging , placement of the vvm if multiple doses of vaccine are packaged together in the same pouch or overwrap , the vvm should be placed on the primary container so that it remains with the vaccine until each dose is delivered . however , in this case the vvm may be obscured by the secondary packaging , which could present challenges for inspection during storage . many of the features discussed here also provide safety and program suitability in low - resource settings , in addition to ease of use . for all new oral vaccine presentations , human factors and usability evaluations should be performed to assess ease of use , correct dosing , and any potential risks or errors resulting from the presentation . one important ergonomic factor for delivery of oral presentations is squeezability of the delivery device , which is determined by container shape , material stiffness , and viscosity of the vaccine . optimal usability and safety are achieved by minimizing the steps ( and subsequent risks ) associated with preparing and delivering the vaccine at the point of use , and by giving the vaccinator fine - tuned control over the dispensing speed . presentations requiring reconstitution or mixing of vaccine components at the point of use increase the time required for and the complexity of the delivery process , and have resulted in errors such as use of incorrect diluents or administration of the diluent alone by practitioners who mistake it for the complete vaccine . to this end , a critical characteristic of the pspq is that oral vaccine formulations be in a ready - to - use format ( no reconstitution needed ) for developing - country , public - sector immunization programs . compliance with this requirement is compulsory , but requests for exceptions are reviewed by the pspq standing committee , taking into account the public health need . if preparation steps are necessary , the mixing process and containers should be easy to use and should minimize the risk of errors , and all required components should be disposable and packaged with each dose of vaccine . this co - packaging requirement applies to water for reconstitution as well , as clean water may not be available at immunization sites in low - resource settings . integrated reconstitution technologies in which multiple liquid or dry vaccine components are packaged within the same primary container and mixed within the device prior to administration can simplify the preparation process and reduce the risk of errors , but can also be costlier . some features that impart ease of use to containers and delivery devices such as ready - to - use formulations also increase safety of use . an inherent safety feature of oral vaccines is that they are administered without needles , and thus needlestick injuries and sharps disposal can be avoided . another safety consideration is the use of packaging and delivery - device designs that reduce the risk of accidental injection of an oral vaccine , which can cause adverse events and render vaccination ineffective . this error has occurred with oral rotavirus vaccines , particularly those in packaging that appear similar to vaccines for injection . the risk of injection precludes use of oral vaccine presentations in glass vials intended for delivery with a syringe with the needle removed . manufacturing feasibility and cost are also important for selecting containers and delivery devices for oral vaccines . for vaccines that may be marketed to adult travelers as well as provided to infants in low - resource settings , different presentations and different final manufacturing processes may be needed . polymer containers may have lower per - unit costs than glass vials and be easier to use , but since manufacturers are likely to have glass - vial - filling facilities in place , using an alternative fill - finish process will entail start - up costs , could require building or repurposing facilities , and might increase a company 's technical burden and risks . for vaccines needing reconstitution , integrated reconstitution devices can simplify delivery but may increase manufacturing complexity and device costs . if a manufacturer is contracting with other companies for packaging or delivery components , having only a single source supplier is a risk , since a problem at this source could hold up the entire production process . immunization program suitability . for example , accurate dose preparation and measurement , as well as simple , controlled vaccine administration are desirable not only for ease of use but for program suitability in low - resource settings where health care workers may have minimal training . use of a polymer squeeze tube as the primary container and delivery device increases safety , addresses ease of use , and reduces packaging volume all of which contribute to program suitability single - dose presentations offer advantages in ease of use , as they do not require measurement of doses ; however , they can increase manufacturing costs and cold chain storage volumes . multi - dose presentations reduce space needed in the cold chain , but they increase vaccine wastage , particularly if the vaccine is at risk of contamination or is not stable for more than one immunization session after opening . tradeoffs such as these must be assessed in determining the optimal primary container for a new vaccine . vaccine manufacturers as well as developers of packaging and administration components can get feedback on the suitability of proposed presentations of vaccines in development from the immunization practices advisory committee 's delivery technologies working group and from the who prequalification group . cost containment . immunization program managers should ideally assess the overall program cost to deliver a vaccine in different presentations , in addition to vaccine price itself . some technologies may increase safety or ease of use , but likely will increase vaccine purchase costs and the burden on the cold chain , so these attributes must be balanced in selection of a vaccine presentation . developers will want to design primary containers and delivery devices with the needs of manufacturers and immunization programs in mind , in order to maximize suitability while minimizing costs . for example , for polymer tubes , a multi - mono - dose configuration in which multiple , conjoined , single - dose containers share a single label and vvm can reduce manufacturing costs and demands on storage space . however , this approach requires a design that prevents users from removing a single container without the label information . in summary , oral vaccines against bacterial pathogens such as etec and shigella that cause diarrhea in millions of children in low - resource countries could save lives and reduce the burden of serious illnesses . but new oral enteric vaccine candidates must be buffered against stomach acid , formulated in minimal dose volumes , filled into containers that protect antigens and adjuvants but minimize the load on the cold chain , and can be safely administered in the correct dose by minimally trained vaccinators working in challenging environments all within the constraints of manufacturability and attention to cost . product designers who take these features into account in their development processes will be able to position new oral vaccines for implementation in routine immunization programs , and these programs in turn will achieve optimal vaccine impact . this work was funded in whole by a grant from the bill & melinda gates foundation . the views expressed herein are solely those of the authors and do not necessarily reflect the views of the foundation .
abstractoral administration of vaccines is simpler and more acceptable than injection via needle and syringe , particularly for infants ( fig . 1 ) this route is promising for new vaccines in development against enterotoxigenic escherichia coli ( etec ) and shigella that cause childhood diarrhea with devastating consequences in low - resource countries . however , vaccine antigens and adjuvants given orally need buffering against the degradative effects of low stomach ph , and the type and volume of antacid buffer require special attention for infants . in addition , container / closure systems must be compatible with vaccine formulations , protect against water and gas transfer , and have minimal impact on the cold chain . health care workers in demanding low - resource settings need an administration device that is easy to use , yet will accurately measure and safely deliver the correct vaccine dose . developers must consider manufacturing capabilities , and immunization program managers want affordable vaccines . as new combination enteric vaccine candidates advance into clinical evaluation , features of the final vaccine presentation liquid or dry format , diluent , buffer , primary and secondary packaging , and administration device should be taken into account early in product development to achieve the greatest possible impact for the vaccine .
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Proceed to summarize the following text: the clinical use of hiv protease inhibitors ( pis ) for the treatment of hiv infection has significantly improved survival of patients ; so , pis have become a key component in the highly active antiretroviral therapy ( haart ) . meanwhile , hiv infection and treatment with haart have been associated with a high incidence of endothelial dysfunction and cardiovascular disease including pulmonary arterial hypertension [ 13 ] . although the mortality associated with hiv-1 infection has been markedly reduced with the advent of haart , cardiovascular complications have emerged as a major non - infectious cause of death in hiv-1 patients . long - term haart may promote oxidative stress and vascular dysfunction leading to cardiovascular complications . we have recently demonstrated that ritonavir ( rtv ) , one of hiv pis , causes endothelial dysfunction in porcine coronary and pulmonary arteries and in human endothelial cells , associated with a decrease in endothelial nitric oxide synthase ( enos ) expression and increased oxidative stress [ 58 ] . endothelial dysfunction is the initial step in pathogenesis of the vascular lesion formation and is characterized by decreased bioavailability of nitric oxide ( no ) ; this may be due to enhanced no catabolism secondary to increased superoxide anion ( o2 ) production or reduced expression and/or activity of enos . therefore , using antioxidants that directly scavenge o2 or inhibit its production in cells may effectively reduce the cellular level of oxidative stress . nordihydroguaiaretic acid ( ndga ) is a natural herbal antioxidant found in the creosote bush , larrea tridentate . the objective of this study was to investigate whether ndga can protect vascular tissues against rtv - induced vascular injury . we used a clinically relevant concentration of rtv as well as low concentrations of ndga . porcine pulmonary arteries were used to study the effect of rtv and ndga on vasomotor functions , enos expression , and o2 production . human pulmonary artery endothelial cells ( hpaecs ) were also used to elucidate changes in enos expression and oxidative stress . this study may provide valuable information leading to the development of effective strategies for the prevention and treatment of cardiovascular and pulmonary vascular complications in hiv - infected patients . thrombaxane a2 analogue u46619 ( 9,11-dideoxy-11 , 9-epoxymethanoprosta - glandin f2 ) , bradykinin , sodium nitroprusside ( snp ) , phosphate buffered saline ( pbs ) solution were obtained from sigma chemical co. rtv was obtained from aids research and reference reagent program ( nih ) . dulbecco s modified eagle s medium ( dmem ) was obtained from life technologies , inc . porcine lungs from freshly slaughtered young adult farm pigs were harvested and placed in cold pbs solution ; the main pulmonary arteries were flushed with pbs to prevent clot formation , and the lungs were immediately transported to the laboratory on ice . the pulmonary arteries were cleared of surrounding connective tissue and dissected from the middle and upper lobes . the vessels were cut into 5 mm rings and incubated in culture medium ( dmem with 1% antibiotic solution ) for 24 hours at 37c and 5% co2 with one of the following treatments : 15 mol / l rtv alone or both ndga ( 1.75 or 3.5 dmem with 0.1% dimethylsulfoxide ( dmso ) served as a control because rtv and ndga were dissolved in dmso . the myograph tension system used in our laboratory has been previously described [ 58,13 ] . briefly , after 24 hours of incubation in culture medium in the treatment groups described above , the vessel rings were suspended between the wires of the organ bath myograph chamber ( danish myo technology organ bath 700 mo , aarhus denmark ) in 6 ml kreb s solution at 37c and bubbled with pure oxygen . the rings were subjected to a slow , stepwise increase in tension until they reached 3 mn . after equilibration , each ring was then precontracted with 20 l u46619 ( 10 mol / l ) . after 60 minutes of contraction , the relaxation was generated by adding 60 l of bradykinin at concentrations of 10 when cells grew to 80 to 90% confluence in six - well culture plates , they were treated with dmso ( control ) or rtv with and without ndga at the concentrations described above for 24 hours at 37c . cells were then applied to studies of enos expression and superoxide anion production . the porcine pulmonary artery endothelial cells were collected by scraping the luminal surface of cultured artery rings with surgical blades . total cellular rna was then extracted using an rnaqueous-4pcr kit ( ambion , austin , tx ) , and enos mrna levels were detected by real - time pcr . primers for enos were designed via the beacon designer 2.1 software ( bio - rad , hercules , ca ) as previously reported . relative mrna levels of enos were normalized to cd31 mrna levels for porcine vessels , and to glyceraldehydes 3-phosphate dehydrogenase ( gapdh ) mrna levels for hpaecs and presented as 2 as previously described . porcine pulmonary artery rings treated with rtv and/or ndga were fixed in 10% formalin and embedded in paraffin . samples were then sectioned at a 5-m thickness and slides were incubated with monoclonal antibody against human enos ( 1:500 ) for 30 min at room temperature . after being washed with pbs , an avidin - biotin reaction using peroxidase enzyme was used for protein detection ( abc kit ; vector laboratories , burlingham , ca ) . images were captured using microscopy as previously described . levels of o2 produced by the endothelial cells of porcine arteries were detected using the lucigenin - enhanced chemiluminescence method previously described in our studies . the data were represented in relative light units per second ( rlu / second ) for each sample . to normalize the data for each sample , the area of each vessel segment was precisely measured using a digital caliper . glutathione ( gsh ) is an intracellular antioxidant , which reversely correlates with cellular ros levels . hpaecs were treated with either rtv ( 15 mol / l ) or pretreated with ndga for 30 min followed by rtv treatment for 45 min . cellular gsh levels were measured as per manufacture s instructions by following a gsh - glo glutathione assay kit ( promega , madison , wi ) . thrombaxane a2 analogue u46619 ( 9,11-dideoxy-11 , 9-epoxymethanoprosta - glandin f2 ) , bradykinin , sodium nitroprusside ( snp ) , phosphate buffered saline ( pbs ) solution were obtained from sigma chemical co. rtv was obtained from aids research and reference reagent program ( nih ) . dulbecco s modified eagle s medium ( dmem ) was obtained from life technologies , inc . porcine lungs from freshly slaughtered young adult farm pigs were harvested and placed in cold pbs solution ; the main pulmonary arteries were flushed with pbs to prevent clot formation , and the lungs were immediately transported to the laboratory on ice . the pulmonary arteries were cleared of surrounding connective tissue and dissected from the middle and upper lobes . the vessels were cut into 5 mm rings and incubated in culture medium ( dmem with 1% antibiotic solution ) for 24 hours at 37c and 5% co2 with one of the following treatments : 15 mol / l rtv alone or both ndga ( 1.75 or 3.5 dmem with 0.1% dimethylsulfoxide ( dmso ) served as a control because rtv and ndga were dissolved in dmso . the myograph tension system used in our laboratory has been previously described [ 58,13 ] . briefly , after 24 hours of incubation in culture medium in the treatment groups described above , the vessel rings were suspended between the wires of the organ bath myograph chamber ( danish myo technology organ bath 700 mo , aarhus denmark ) in 6 ml kreb s solution at 37c and bubbled with pure oxygen . the rings were subjected to a slow , stepwise increase in tension until they reached 3 mn . after equilibration , each ring was then precontracted with 20 l u46619 ( 10 mol / l ) . after 60 minutes of contraction , the relaxation was generated by adding 60 l of bradykinin at concentrations of 10 when cells grew to 80 to 90% confluence in six - well culture plates , they were treated with dmso ( control ) or rtv with and without ndga at the concentrations described above for 24 hours at 37c . the porcine pulmonary artery endothelial cells were collected by scraping the luminal surface of cultured artery rings with surgical blades . total cellular rna was then extracted using an rnaqueous-4pcr kit ( ambion , austin , tx ) , and enos mrna levels were detected by real - time pcr . primers for enos were designed via the beacon designer 2.1 software ( bio - rad , hercules , ca ) as previously reported . relative mrna levels of enos were normalized to cd31 mrna levels for porcine vessels , and to glyceraldehydes 3-phosphate dehydrogenase ( gapdh ) mrna levels for hpaecs and presented as 2 as previously described . porcine pulmonary artery rings treated with rtv and/or ndga were fixed in 10% formalin and embedded in paraffin . samples were then sectioned at a 5-m thickness and slides were incubated with monoclonal antibody against human enos ( 1:500 ) for 30 min at room temperature . after being washed with pbs , an avidin - biotin reaction using peroxidase enzyme was used for protein detection ( abc kit ; vector laboratories , burlingham , ca ) . levels of o2 produced by the endothelial cells of porcine arteries were detected using the lucigenin - enhanced chemiluminescence method previously described in our studies . the data were represented in relative light units per second ( rlu / second ) for each sample . to normalize the data for each sample , the area of each vessel segment was precisely measured using a digital caliper . glutathione ( gsh ) is an intracellular antioxidant , which reversely correlates with cellular ros levels . hpaecs were treated with either rtv ( 15 mol / l ) or pretreated with ndga for 30 min followed by rtv treatment for 45 min . cellular gsh levels were measured as per manufacture s instructions by following a gsh - glo glutathione assay kit ( promega , madison , wi ) . the effects of rtv and ndga on vasomotor functions in porcine pulmonary arteries were determined using a myograph system which included measurement of vascular contraction ( induced by thromboxane a2 analog u46619 ) , endothelium - dependent relaxation ( bradykinin ) and endothelium - independent relaxation ( snp ) . porcine pulmonary artery rings ( 5 per group ) were cultured for 24 hours in dmem with dmso as a control or treated with a clinically relevant amount of rtv alone ( 15 mol / l ) or rtv and ndga ( 1.75 or 3.50 mol / l ) . in response to u46619 ( 10 m ) , the control vessels contracted maximally to 25 mn . treatment with rtv reduced contraction by 36% compared with controls ; when cultured together with ndga ( 1.75 and 3.50 mol / l ) , the contraction increased by 21% and 71% , respectively , compared with rtv - alone group ( figure 1a , p<0.05 , n=5 ) . endothelium - dependent vasorelaxation was induced by the addition of brandykinin ( 10 mol / l ) to the organ bath , and the control vessels relaxed by 36% , while rtv - treated vessels relaxed by only 22% , which represented a 39% reduction in endothelium - dependent vasorelaxation ; when co - cultured with ndga ( 1.75 and 3.50 mol / l ) , the relaxation increased by 25% and 48% , respectively , over the relaxation achieved by the rtv - treated vessels ( figure 1b , p<0.05 , n=5 ) . in response to snp , treatment with rtv reduced endothelium - independent vasorelaxation by 17.5% ; when ndga was included with the rtv treatment , endothelium - independent relaxation was restored ( figure 1c , p<0.05 , n=5 ) . taken together , these data demonstrate that rtv induces vasomotor dysfunction in porcine pulmonary arteries , and ndga effectively inhibits it . in our previous studies , we demonstrated that enos protein is involved in rtv - induced vasomotor dysfunction in porcine pulmonary arteries ; rtv treatment significantly decreases enos expression in a concentration - dependent manner in both porcine pulmonary artery cells and hpaecs . in this study after treatment with 15 mol / l of rtv , there was a reduction in enos mrna levels by 21% in arterial rings and by 38.5% in hpaecs compared with their respective dmso controls . however , after co - treatment with rtv and ndga ( 1.75 and 3.50 mol / l ) , enos mrna levels in the vessel rings and hpaecs significantly increased compared with rtv only group ( figure 2a , b , p<0.05 , n=3 ) . enos protein levels in the porcine pulmonary arteries were analyzed using immunohistochemistry staining ( figure 3 ) . levels of the dark brown - staining enos protein were reduced at the luminal endothelial layer in the rtv alone group compared with the dmso control group . when co - treated with ndga and rtv , enos immunoreactivity was significantly increased to the control level ; meanwhile ndga alone did not alter the enos protein level compared with controls . therefore , ndga inhibits the rtv - induced decrease of enos expression at the protein level in porcine pulmonary arteries . the hiv protease inhibitor rtv has been shown to significantly increase superoxide anion production in porcine pulmonary arteries . to investigate whether ndga can block the superoxide production induced by rtv , superoxide anion production in porcine pulmonary arterial rings and hpaecs was analyzed using lucigenin - enhanced chemiluminescence and gsh - mediated quantification of ros , respectively . superoxide anion levels in the arterial rings were significantly increased in response to rtv treatment . rtv ( 15 mol / l ) treatment of the vessel rings increased the chemiluminescence signal to 8.551.32 rlu / sec / mm , compared with the control value of 5.660.66 rlu / sec / mm ( figure 4a , p<0.05 , n=4 ) . this indicates a 51% increase of superoxide anion production in the rtv - treated rings over controls . co - treatment with ndga ( 3.5 mol / l ) in addition to rtv led to a reduction in the chemiluminescence signal to 6.361.07 rlu / sec / mm , indicating a partial reversal of the effects of rtv to increase ros . rtv treatment led to a significant decrease in gsh levels by 56% in hpaecs compared to controls ( figure 4b , p<0.05 , n=4 ) ; this indicates that ros production increased by 56% . as before , this rtv - induced increase in ros , was blocked by ndga . in this case , the ndga alone group showed a higher gsh level than the control group , indicating its capacity to further reduce ros levels over baseline control levels . the effects of rtv and ndga on vasomotor functions in porcine pulmonary arteries were determined using a myograph system which included measurement of vascular contraction ( induced by thromboxane a2 analog u46619 ) , endothelium - dependent relaxation ( bradykinin ) and endothelium - independent relaxation ( snp ) . porcine pulmonary artery rings ( 5 per group ) were cultured for 24 hours in dmem with dmso as a control or treated with a clinically relevant amount of rtv alone ( 15 mol / l ) or rtv and ndga ( 1.75 or 3.50 mol / l ) . in response to u46619 ( 10 m ) , the control vessels contracted maximally to 25 mn . treatment with rtv reduced contraction by 36% compared with controls ; when cultured together with ndga ( 1.75 and 3.50 mol / l ) , the contraction increased by 21% and 71% , respectively , compared with rtv - alone group ( figure 1a , p<0.05 , n=5 ) . endothelium - dependent vasorelaxation was induced by the addition of brandykinin ( 10 mol / l ) to the organ bath , and the control vessels relaxed by 36% , while rtv - treated vessels relaxed by only 22% , which represented a 39% reduction in endothelium - dependent vasorelaxation ; when co - cultured with ndga ( 1.75 and 3.50 mol / l ) , the relaxation increased by 25% and 48% , respectively , over the relaxation achieved by the rtv - treated vessels ( figure 1b , p<0.05 , n=5 ) . in response to snp , treatment with rtv reduced endothelium - independent vasorelaxation by 17.5% ; when ndga was included with the rtv treatment , endothelium - independent relaxation was restored ( figure 1c , p<0.05 , n=5 ) . taken together , these data demonstrate that rtv induces vasomotor dysfunction in porcine pulmonary arteries , and ndga effectively inhibits it . in our previous studies , we demonstrated that enos protein is involved in rtv - induced vasomotor dysfunction in porcine pulmonary arteries ; rtv treatment significantly decreases enos expression in a concentration - dependent manner in both porcine pulmonary artery cells and hpaecs . in this study after treatment with 15 mol / l of rtv , there was a reduction in enos mrna levels by 21% in arterial rings and by 38.5% in hpaecs compared with their respective dmso controls . however , after co - treatment with rtv and ndga ( 1.75 and 3.50 mol / l ) , enos mrna levels in the vessel rings and hpaecs significantly increased compared with rtv only group ( figure 2a , b , p<0.05 , n=3 ) . enos protein levels in the porcine pulmonary arteries were analyzed using immunohistochemistry staining ( figure 3 ) . levels of the dark brown - staining enos protein were reduced at the luminal endothelial layer in the rtv alone group compared with the dmso control group . when co - treated with ndga and rtv , enos immunoreactivity was significantly increased to the control level ; meanwhile ndga alone did not alter the enos protein level compared with controls . therefore , ndga inhibits the rtv - induced decrease of enos expression at the protein level in porcine pulmonary arteries . the hiv protease inhibitor rtv has been shown to significantly increase superoxide anion production in porcine pulmonary arteries . to investigate whether ndga can block the superoxide production induced by rtv , superoxide anion production in porcine pulmonary arterial rings and hpaecs was analyzed using lucigenin - enhanced chemiluminescence and gsh - mediated quantification of ros , respectively . superoxide anion levels in the arterial rings were significantly increased in response to rtv treatment . rtv ( 15 mol / l ) treatment of the vessel rings increased the chemiluminescence signal to 8.551.32 rlu / sec / mm , compared with the control value of 5.660.66 rlu / sec / mm ( figure 4a , p<0.05 , n=4 ) . this indicates a 51% increase of superoxide anion production in the rtv - treated rings over controls . co - treatment with ndga ( 3.5 mol / l ) in addition to rtv led to a reduction in the chemiluminescence signal to 6.361.07 rlu / sec / mm , indicating a partial reversal of the effects of rtv to increase ros . rtv treatment led to a significant decrease in gsh levels by 56% in hpaecs compared to controls ( figure 4b , p<0.05 , n=4 ) ; this indicates that ros production increased by 56% . as before , this rtv - induced increase in ros , was blocked by ndga . in this case , the ndga alone group showed a higher gsh level than the control group , indicating its capacity to further reduce ros levels over baseline control levels . the major finding of this study is that the natural herbal antioxidant ndga can effectively inhibit the reduction in vasocontractility and endothelium - dependent vasorelaxation induced by hiv protease inhibitor rtv in porcine pulmonary arteries . this may be mediated by restoration of enos expression , as we found ndga blocked the rtv - induced decrease of enos expression in both porcine pulmonary arteries and hpaecs . furthermore , ndga inhibits rtv - induced superoxide anion production in porcine pulmonary arteries and hpaecs . as such , this study suggests that ndga may be a useful therapy in hiv - infected patients . the endothelium is considered an active biologic interface between the blood and all other tissues . functions of the endothelium include control of vascular tone , modulation of inflammation , promotion or inhibition of vascular growth , and regulation of thrombosis . endothelial dysfunction can be broadly defined as an imbalance between vasodilating and vasoconstricting substances produced by the endothelium . in addition , it has been seen in a variety of pathological conditions such as atherosclerosis , hypercholesterolemia , diabetes , hypertension , heart failure , cigarette smoking and aging . it is characterized by decreased bioavailability of no , which may be due to enhance no catabolism secondary to increased superoxide anion production or reduced expression and/or activity of enos . in this study , we used a clinically relevant concentration of rtv ( 15 mol / l ) and a well - defined myograph tension system to test our hypothesis that the natural herbal antioxidant ndga can protect vasomotor function against oxidative damage in porcine pulmonary arteries . our data demonstrate that rtv significantly decreases endothelium - dependent vasorelaxation in response to bradykinin in porcine pulmonary arteries , as expected . these changes indicate that rtv has direct effects on the vascular endothelium and are consistent with several clinical studies . meanwhile , when the pulmonary artery rings were cultured with ndga ( 1.75 or 3.50 mol / l ) in addition to rtv , there was a significant reversal of the detrimental effects of rtv on vasomotor reactivity in a concentration - dependent manner . in other studies , natural antioxidants such as ginsenosides , ginkgo , soybean isoflavonoid equol , capsaicin , and dihydroxybenzyl alcohol have been shown to block rtv - induced endothelial dysfunction . like these antioxidants , ndga showed a strong effect against rtv - induced vasomotor dysfunction . in the endothelium , no is constitutively generated from the conversion of l - arginine to l - citrulline by the enzymatic action of enos . however , enos may become dysfunctional or its expression may decrease under various pathological conditions . dysfunctional enos or low levels of enos reduce no availability , which results in impaired endothelium - dependent vasorelaxation and accelerated vascular lesion formation ; the development of pulmonary arterial hypertension is likewise promoted . in the current study , real - time pcr analysis showed that there was reduced enos mrna expression in the rtv - treated porcine pulmonary arteries and cultured hpaecs , and immunohistochemistry showed a significant decrease of the enos protein levels in the endothelium of rtv treated vessels . however , ndga effectively blocked these effects of rtv on downregulating enos at both the mrna and protein levels , suggesting a potential therapeutic role for ndga . in a previous paper , it was reported that the two haart drugs rtv and azt substantially increased the phosphorylation of erk2 in hpaecs , and a mek / erk inhibitor effectively blocked rtv- or azt - induced reductions in enos protein levels in hpaecs ; this indicated that mapk signaling is involved in the development of endothelial dysfunction induced by haart drugs . in this study , we found that ndga reversed the decrease of enos protein level ; this may be due to inhibition of the erk 1/2 activation induced by rtv . , it has been reported that treatment with ndga reduces erk 1/2 phosphorylation in 15-lox-1 overexpressing hct-116 cells . our previous studies have demonstrated that some haart drugs including rtv significantly increase superoxide anion production in porcine pulmonary arteries and hpaecs . increased ros contribute to endothelial dysfunction and vascular pathology . superoxide can influence no bioavailability by binding to no to form the highly reactive intermediate onoo . the speed of this reaction is about 10 times faster than the dismutation of superoxide by the superoxide dismutase . furthermore , increased superoxide and onoo may impair the function of enos by reducing the bioavailability of its coenzyme tetrahydrobiopterin . onoo may also influence the synthesis of other endothelial mediators , such as the enzyme prostacyclin synthase , therefore leading to reduced production of vasodilator , anti - aggregant and anti - inflammatory prostaglandins . our study demonstrates that ndga also effectively abolishes the oxidative stress induced by rtv in both porcine pulmonary arteries and hpaecs ; ndga either blocks the superoxide anion production or directly scavenges superoxide anion . the latter is more likely , considering the ndga alone treatment group led to a higher gsh level than the control group , indicating a lower superoxide anion level . also , it has been reported that ndga is a strong superoxide anion scavenger by floriano - sanchez . in summary , the natural herbal compound ndga could potentially have a protective role against rtv - induced vascular dysfunction . ndga effectively inhibits the detrimental effects of hiv protease inhibitor rtv on the vasomotor functions of endothelial and smooth muscle cells in porcine pulmonary arteries . ndga also blocks the decrease in enos expression and the increase in superoxide anion production induced by rtv . these findings suggest that ndga may have clinical applications in the prevention of systemic and pulmonary vascular complications associated with the long term use of haart drugs in hiv patients .
summarybackgroundhiv infection and treatment with highly active antiretroviral therapy ( haart ) including hiv protease inhibitor ritonavir ( rtv ) have been associated with endothelial dysfunction and cardiovascular disease including pulmonary arterial hypertension . the objective of this study was to determine if nordihydroguaiaretic acid ( ndga ) , a natural herbal antioxidant found in the creosote bush larrea tridentate , can protect vascular tissues against rtv - induced vascular injury.material/methodsfresh porcine pulmonary artery ( pa ) rings were treated with a clinically relevant concentration of rtv ( 15 mol / l ) with or without ndga for 24 hours , and then subjected to myograph analysis for vasomotor reactivity . expression of endothelial nitric oxide synthase ( enos ) in both treated pa rings and human pulmonary artery endothelial cells ( hpaecs ) was analyzed by real - time pcr and immunohistochemistry . oxidative stress levels were analyzed with the lucigenin - enhanced chemiluminescence and glutathione assay.resultsin response to bradykinin at 1010 mol / l , rtv - treated pa rings showed a 39% reduction in endothelium - dependent vasorelaxation compared with the control vessels ( p<0.05 ) ; when co - cultured with ndga ( 1.75 or 3.50 mol / l ) , the relaxation increased by 25% and 48% , respectively . rtv also decreased the maximal contraction and endothelium - independent vasorelaxation in rtv - treated vessels , while ndga improved these vasomotor responses . in addition , treatment of rtv significantly decreased enos mrna levels in both porcine pas and hpaecs , and reduced enos immunoreactivity in porcine pas , while ndga significantly inhibited this effect of rtv . furthermore , ndga significantly blocked rtv - induced increase of superoxide anion in the pa rings and inhibited rtv - induced decrease of glutathione in hpaecs.conclusionsndga effectively inhibits the detrimental effects of hiv protease inhibitor rtv on vasomotor functions in porcine pas . ndga also blocks rtv - induced decrease of enos expression and increase of oxidative stress in both porcine pas and hpaecs . this study may provide valuable information for the development of effective strategies for the prevention and treatment of haart - associated cardiovascular complications .
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Proceed to summarize the following text: de novo human artificial chromosomes ( hac ) are small , extrachromosomal elements that contain a functional centromere , enabling their correct replication and segregation as stable normal chromosomes in human cells ( kouprina et al . 2014 ; grimes and monaco 2005 ) , together with the endogenous chromosomes . de novo hac are generated by introducing specific sequences such as -satellite ( alphoid ) dna into eukaryotic cells . the essential requirement for a functional centromere formation in hac is -satellite dna , containing higher - order repeat sequences and a centromere protein b binding sequence ( cenp - b box ) ( bergmann et al . 2012 ) . by mechanisms not yet elucidated , the cell is able to recognise the transgenic dna as centromeric and seed the deposition of specific protein / epigenetic markers on the exogenous molecules , leading to the formation of a fully active centromere , thus transforming the episomal vector into a full - fledged artificial chromosome ( grimes and monaco 2005 ) . in human cells , autonomous hac are characterised by high mitotic stability ( grimes and monaco 2005 ; moralli et al . 2009 ) , unlike other viral vectors which integrate into the host chromosome and may result in insertional mutagenesis ( baum 2007 ) . as there is no limit on the size of the dna fragments that can be incorporated into hac , they represent ideal vectors for the delivery of large genomic dna regions containing genes and their corresponding regulatory elements . hac containing the entire human genomic hypoxanthine phosphoribosyltransferase ( hprt ) region fully complemented the hprt deficiency in cultured human fibrosarcoma ht1080 cells ( meja et al . the de novo hac structure was relatively simple compared to normal chromosomes ( meja et al . known methods for the delivery of large pieces of dna are inefficient , often resulting in dna shearing and degradation , which is a major obstacle in developing a hac expression system in different cell types . current methods of introducing dna into cells include the use of lipofecting agents , electroporation and viral and non - viral vectors . each of these methods shares the problem that only small pieces of dna can be put into cells without the dna being damaged . large dna fragments have very low transfection efficiencies or break up when entering cells using current methods . this has made it very difficult in the past to get sufficiently large pieces of dna into cells to form a hac . in order to use a hac for gene therapy , most gene expression studies in human embryonic stem cells ( hesc ) utilise lentiviral , adenoviral and adeno - associated ( aav ) viral vectors for gene delivery . however , lentiviral vectors integrate randomly at multiple sites within the host genome leading to insertional mutagenesis , and although adenoviral vectors remain episomal , silencing post - transduction may occur . another disadvantage is that the capacity of aav and lentiviral vectors is limited to approximately 5 and 10 kb of dna , respectively . the herpes simplex virus type 1 ( hsv-1)-based amplicon system offers enormous potential as a versatile tool for gene delivery and has several unique features that distinguish it from rest of the viral vector systems and chemical - mediated gene transfer methods . it can accommodate and deliver up to 152 kb of exogenous dna into the mammalian cells ( saeki et al . . the amplicon dna does not integrate into the host chromosomes , thus reducing the risk of insertional mutagenesis . the head - to - tail , rolling - circle dna replication mechanism of hsv-1 also allows amplification of amplicon plasmid as concatemers leading to increased copy number of transgenes and consequently their expression . most genes in the hsv-1 viral genome are not essential for virus replication and therefore , can be deleted without affecting the virus production from the cultured cells . the lack of viral genes and the availability of amplicon production in a helper virus - free manner results in limited immunogenic and cytotoxic effects . most importantly , the hsv-1 amplicon can transduce a wide variety of cell types across a wide range of species , including dividing and quiescent cells . the hac vectors are firstly retrofitted with the hsv-1 origin of replication and packaging signal ( fig . 1 ) . infectious particles are then produced by co - transfecting , into the permissive cells such as african green monkey kidney vero 2 - 2 cells , ( a ) the desired amplicon dna ( containing the transgene , the hsv pac signal and hsv oris ) , ( b ) the fhsvpac27 0 + vector containing the complete hsv-1 genome save for the deletion of the pac sequence and the essential gene icp27 , and ( c ) an icp27 expressing plasmid such as pebhicp27 ( saeki et al . a large bac , carrying alpha satellite and/or transgenes , is retrofitted with the hsv-1 necessary elements via loxp - cre recombination with a smaller plasmid carrying the hsv-1 origin of replication and packaging signalfig . 2production of infectious particles . the hsv-1 amplicon hac vector is transfected into helper cells along with the whole hsv-1 genome ( lacking the icp27 gene and the packaging signal ) and a small plasmid expressing the essential hsv-1 protein icp27 . the cell assembles the hsv-1 capsids and packages the hsv-1 amplicon hac vector assembly of hsv-1 amplicon hac vectors . a large bac , carrying alpha satellite and/or transgenes , is retrofitted with the hsv-1 necessary elements via loxp - cre recombination with a smaller plasmid carrying the hsv-1 origin of replication and packaging signal production of infectious particles . the hsv-1 amplicon hac vector is transfected into helper cells along with the whole hsv-1 genome ( lacking the icp27 gene and the packaging signal ) and a small plasmid expressing the essential hsv-1 protein icp27 . the cell assembles the hsv-1 capsids and packages the hsv-1 amplicon hac vector helper functions for the amplification and packaging / cleavage of amplicon dna into hsv-1 virions are supplied in trans by fhsvpac27 0 + and pebhicp27 . however , fhsvpac27 0 + itself can not be packaged because it lacks the packaging signal , the icp27 gene and hsv-1 origin of replication . moreover , its size has been increased by the addition of stuffer elements so that the whole construct no longer fits into the hsv virion , thus further reducing the risk of generating replication - competent helper viruses . the hsv-1 amplicon system , therefore , provides a safe and effective mechanism for gene transfer , as amplicons infect the target cells , but lack the components for generating the wild - type virus . the hsv-1 amplicon dna can accommodate one or more transgene cassettes as long as its total size does not exceed 152 kb . upon provision of hsv helper functions in trans , the amplicon plasmid undergoes head - to - tail rolling circle replication and results in concatemers of dna leading to a total size of 152 kb which is then cleaved at the pac signal and packaged into hsv virions ( fig . 1 ) . the transgenic material is carried directly to the nucleus , intact ( saeki et al . 2003 ; wade - martins et al . this feature of the hsv-1 amplicon delivery system is especially important for the generation of human artificial chromosomes and has allowed us to obtain direct hac formation in human embryonic stem cells for the first time . the generation of hac in hesc and ips cells is an essential step to gene expression and gene therapy studies . cells obtained from a patient may be reprogrammed to form ips cells , then modified ex vivo with a hac vector containing the desired gene and reintroduced into the patient . hesc could be differentiated into immunoglobulin - producing cells , which could prove useful in vaccine production . the hac generated in our laboratory and others , have to date been obtained in tumour - derived or immortalised cell lines , such as the human fibrosarcoma cell line ht1080 . before hac can become fully viable vectors for gene expression studies and potentially gene therapy , it is necessary to study their behaviour in cells that have normal genetic backgrounds such as hesc that can be propagated indefinitely in vitro and can be differentiated in a range of different cell types . in our study , we assembled hsv-1 amplicon technology hac input vectors carrying reporter genes ( mandegar et al . 2011 ) . the vectors were delivered to the hesc cell lines hues-2 and hues-10 ( cowan et al . 2004 ) and the results compared to similar experiments in the human fibrosarcoma ht1080 cells , which are highly efficient at hac formation ( grimes and monaco 2005 ) . the transduction efficiency using hsv-1 amplicons is high , ranging from 27 to 40 % in hesc ( mandegar et al . 2011 ) and up to 70 % in ht1080 cells ( moralli et al . 2006 ) . following g418 selection , several independent clones can be obtained from each vector in both hesc and ht1080 cells . in standard hac generation experiments , clones are expanded to at least the 10-cm dish stage , before chromosomes are harvested and subjected to fluorescence in situ hybridization ( fish ) to determine if the cells contain hacs or integrations . thus , the karyotypic analysis of a large number of hesc clones can be very time - consuming and expensive . for this reason , the development of an improved harvesting technique , which allows to obtain high - quality metaphase spreads from six - well dishes , proved especially useful for our studies ( moralli et al . this method relies on overnight incubation with a low dose of nocodazole as synchronizing agent . the cells are swelled by treatment in a buffered hypotonic , which dramatically improves chromosome spreading . the quality of the resulting metaphase suspension is such that they can be easily analysed by fish or mfish ( fig . 3mfish on hues10 hesc metaphase spread , obtained with an improved chromosome harvesting procedure mfish on hues10 hesc metaphase spread , obtained with an improved chromosome harvesting procedure in our studies , hesc clones were analysed by fish with hac - specific probes ( fig . 4 ) to determine if the input vector had integrated randomly into the host cell genome or if had formed a hac able to segregate independently . while in ht1080 , the majority of clones contained integrations and hac , whereas in the hesc clones , the input dna had formed hac in a large percentage of cells and there was no integrated dna present ( mandegar et al . the metaphase spread has been hybridised with a 17alpha satellite probe ( red signal ) identifying the hac ( arrow ) , along with the endogenous chromosomes 17 . the small inset show the dapi staining of the hac , as a black and white picture fish on hac containing hesc . the metaphase spread has been hybridised with a 17alpha satellite probe ( red signal ) identifying the hac ( arrow ) , along with the endogenous chromosomes 17 . the small inset show the dapi staining of the hac , as a black and white picture the analysis of the hesc hac with antibodies against essential centromeric proteins ( cenp c ) showed that all the hac had formed a true centromere . when grown in the absence of selection , the hac were mitotically stable , displaying a daily loss rate of 0.030.24 % . furthermore , immunostaining and rt - pcr analysis of pluripotency markers ( oct4 , sox2 , nanog , tra-1 - 60 ) indicated that the cells derived from the hac clones remained pluripotent ( mandegar et al . the pluripotency of the cells was further confirmed following directed differentiation of hac - containing cells into neuronal type cells and the development of teratomas with the three germ layers following the formation assay . importantly , the hac were maintained in the neuronal cells following differentiation , as shown by fish with specific probes . compared to transfection , the hsv-1 amplicon provided highly efficient delivery of small and large dna segments to hues-2 cells , and neither transfection nor the hsv-1 amplicon technique was toxic to hues-2 cells , as shown by measuring the average growth rate post - transduction ( mandegar et al . the hsv-1 amplicon technology is a highly efficient delivery method based on transferring exogenous dna packaged in hsv-1 amplicons into cells . the amplicons efficiently deliver large dna containing genes within high - capacity human artificial chromosome vectors into human embryonic stem cells and generate gene expressing hac in hesc cell lines . the lack of integrated dna in hesc following hsv-1 delivery compared to integrated dna following delivery in cultured cells is a significant difference and will lead to further work on understanding the mechanism of hac generation in human cells . the differences in genome stability between stem and tumour cells indicate that hac will be important in gene therapy strategies for monitoring chromosome stability in different cell types . hac vectors are a viable alternative to gene delivery with viral vectors in hesc with the aim of developing a hac - based system for the delivery of therapeutic genes regulated with tissue - specific promoters to human target cells for ex vivo treatment . the development of ex vivo strategies for gene therapy of inherited genetic disorders and addressing problems in the areas of cancer , aging and metabolic disorders will be key targets for hac therapy .
de novo artificial chromosomes expressing genes have been generated in human embryonic stem cells ( hesc ) and are maintained following differentiation into other cell types . human artificial chromosomes ( hac ) are small , functional , extrachromosomal elements , which behave as normal chromosomes in human cells . de novo hac are generated following delivery of alpha satellite dna into target cells . hac are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation . they are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long - term expression studies in cells unlike other viral gene delivery vectors currently used . transferring the essential dna sequences for hac formation intact across the cell membrane has been challenging for a number of years . a highly efficient delivery system based on hsv-1 amplicons has been used to target dna directly to the es cell nucleus and hac stably generated in human embryonic stem cells ( hesc ) at high frequency . hac were detected using an improved protocol for hesc chromosome harvesting , which consistently produced high - quality metaphase spreads that could routinely detect hac in hesc . in tumour cells , the input dna often integrated in the host chromosomes , but in the host es genome , it remained intact . the hesc containing the hac formed embryoid bodies , generated teratoma in mice , and differentiated into neuronal cells where the hac were maintained . the hac structure and chromatin composition was similar to the endogenous hesc chromosomes . this review will discuss the technological advances in hac vector delivery using hsv-1 amplicons and the improvements in the identification of de novo hac in hesc .
You are an expert at summarizing long articles. Proceed to summarize the following text: first , they must remain quiescent throughout the lifetime of an organism until they are recruited for regeneration , when they must respond to the appropriate cues and reenter the cell cycle . second , once they enter the cell cycle , they must properly balance self - renewal with commitment to differentiation to maintain tissue homeostasis . excess self - renewal leads to over proliferation ( tumor formation ) , whereas excess commitment leads to depletion of stem cell reserves and a reduction in cell number ( degeneration ) . embryonic stem cells ( escs ) do not exit the cell cycle and are only concerned with the second decision . as stem cells commit to differentiation pathways during embryogenesis , lineage options become increasingly restricted , and cells can respond in very different ways to the same signaling cues even though all cells have a full complement of genetic information . this change in cellular competence can be mediated by changes in the intracellular environment ( grimm and gurdon , 2002 ) , changes in transcriptional feedback loops , or epigenetic / chromatin changes such as histone composition or dna methylation ( steinbach et al . , 1997 ; kundu and peterson , 2009 ; kaufman and rando , 2010 ) . no single mechanism is involved in all cell fate transitions , and there are likely to be additional as yet undiscovered mechanisms . although not as well established as the roles of transcription factors ( egli et al . , 2008 ) and histone modifications ( spivakov and fisher , 2007 ) , recent observations link 3d organization of chromatin to gene expression . it is now well accepted that the nucleus is structurally and functionally compartmentalized to favor transcription in specific subnuclear neighborhoods ( lanctt et al . , 2007 ; lieberman - aiden et al . , 2009 ; hakim et al . , 2010 ; laster and kosak , 2010 ) . , 2009 ; peric - hupkes et al . , 2010 ) and the nucleolus ( nmeth et al . , 2010 ) are less - favorable locations for transcription of developmentally regulated genes . in addition , the 3d organization of chromatin can change dramatically as stem cells undergo lineage commitment ( meshorer and misteli , 2006 ) , and this can move specific genes into different compartments ( hiratani et al . , 2010 ) . importantly , once established , structural constraints limit chromatin mobility throughout the remainder of the cell cycle ( chubb et al . , 2002 ; walter et al . , 2003 ) so that large - scale 3d architecture is considerably less plastic than many other features of chromatin structure and could provide a scaffold to preserve a memory of cellular lineage . thus , 3d genome architecture has attracted a great deal of interest to those interested in understanding stem cells and the reprogramming of cellular fates . replication timing has been indirectly linked to chromosome architecture since the 1960s , when it was discovered that the inactive x chromosome in female mammals takes on a compact structure localized to the periphery of the nucleus , known as the barr body , which is coincident with a switch to late replication ( hiratani and gilbert , 2010 ) . as discussed , dna replication takes place in spatially distinct compartments of the nucleus at different times during s phase , and it is now generally appreciated that facultative heterochromatin , including silent gene families ( genes that lack poised polymerase ii ) and imprinted gene loci , are late replicating and generally localized closer to the nuclear periphery or nucleoli . recently , a compelling genome - wide alignment was found between replication timing and the density of chromatin interactions within the mammalian nucleus , and spatial models of how chromatin folds within the nucleus based upon chromatin interaction maps resemble independently derived models of how chromatin is organized into spatially and temporally discrete replication domains ( ryba et al . , 2010 ) . these data provide strong support for the hypothesis that replication domains are self - interacting structural and functional units of chromosomes . implicit in this finding is that genome - wide replication timing profiles provide a clear and measurable property with which to evaluate higher order chromosome structure and function and can rapidly identify regions of chromosomes that undergo remodeling of these architectural features during development or in specific diseases . for example , a comprehensive survey of 10 stages of mouse embryogenesis revealed changes across half of the mouse genome mediated in units of 400800 kb , including a set of down - regulated genes that switch from early to late replicating during loss of pluripotentcy ( hiratani et al . , 2010 ) . these regions remain late replicating throughout the rest of development and are difficult to reprogram back to an embryonic like state ; e.g. , they fail to reestablish esc - specific replication timing and transcription in partially reprogrammed induced pluripotent stem cells ( ipscs ) . it follows that these regions undergo higher - order remodeling events that are difficult to reverse . in fact , these same changes were coincident with chromatin compaction and movement of these regions toward the nuclear periphery ( hiratani et al . , 2010 ) . what regulates these events ? this remains a mystery , but we do know that replication timing is reestablished in each cell cycle by events that take place while chromatin is reorganized after mitosis ( dimitrova and gilbert , 1999 ; lu et al . , 2010 ) . when nuclei from cells staged at different times in the cell cycle are introduced into a cell - free replication initiation system , chromatin in early g1 or g2 phase nuclei replicates in a random temporal order , whereas chromatin from cells in mid to late g1 phase replicates in the cell type characteristic temporal sequence ( lu et al . , 2010 ) . the interpretation of these results is that determinants of replication timing are established during a short 1-h interval of early g1 ( tdp ) and are then lost during replication . importantly , before the tdp , early- and late - replicating chromatin are mobile and irregularly positioned but become anchored and organized in 3d space within the same short time interval as the tdp ( dimitrova and gilbert , 1999 ; chubb et al . , 2002 ; walter et al . , 2003 ; lu et al . , 2010 these studies have suggested a model in which chromatin regulators are concentrated into spatially separated subnuclear compartments by the anchorage and folding of chromosomal domains at the tdp ( gilbert , 2001 ) . in this view , subnuclear compartmentalization is required to establish some type of chromatin mark that is then erased during replication and requires a round of mitosis to reestablish . one possibility is that the factors regulating replication timing set thresholds for the accessibility of s phase promoting factors to replication origins or replicon clusters ( gilbert , 2001 ; thomson et al . , 2010 ) . in fact , larger coordinately replicating domains are found to change replication timing in smaller , 400800-kb increments during differentiation ( hiratani et al . , 2008 ; moreover , the regions between adjacent early- and late - replicating domains traverse spatially segregated compartments of the nucleus and are suppressed for origin activity so that a single unidirectional replication fork duplicates the intervening dna , often over the course of several hours ( ryba et al . interestingly , a histone modification signature exists at the early - replicating boundaries of these intervening timing transition regions ( ryba et al . , 2010 ) , and it is known that at least some of these same chromatin marks are erased at the replication fork and reestablished after the following mitosis ( jansen et al . , 2007 ; aoto et al . , 2008 ; scharf et al . , 2009 ) . however , there are some aspects of higher order chromatin architecture that are clearly not necessary to maintain replication timing once chromatin compartments are established . chromatin within nuclei isolated from quiescent cells , which show measurable changes in global chromatin architecture including massive decondensation of selected regions , retained replication timing determinants ( rtds ; lu et al . , 2010 ) . the tdp may be defined by the establishment of a replication timing program , but the close linkage between replication timing and chromosome architecture implies that important organizational events that determine the folding of chromatin , its degree of compaction , and the potential interactions between genomic segments are also established at this time . in considering causality , it makes little sense that replication timing would be the driving force behind chromatin architecture because this would mean that a chromatin architecture would be established for a replication program that might not be played out if the cells subsequently exit the cell cycle . moreover , it is not obvious why the genome would need a cell type specific temporal sequence to carry out the housekeeping function of genome replication . specific factors present during passage through the tdp , and the replication timing program is a reflection of that architecture . however , that does not imply that replication timing does not influence architecture because different types of chromatin are assembled at different times during s phase ( lande - diner et al . , 2009 ) , so changes in replication timing could rapidly and simultaneously influence the structure of chromatin across large segments of chromosomes , which could profoundly affect the structure assembled in the next cell cycle . i would like to propose that the tdp may represent an as of yet unappreciated window of opportunity for extracellular cues to reprogram chromatin interactions and subnuclear position during cell fate transitions ( fig . once established , these features of chromatin architecture may not be changeable until the next tdp . consistent with this hypothesis , experiments in which ectopically inserted laco sequences were artificially targeted to the nuclear periphery with laci fusions to inner nuclear envelope proteins demonstrated that the targeting protein is not sufficient to reposition chromatin ; repositioning requires mitosis . repositioning silenced some genes in the vicinity of the laco , but the targeting was probabilistic ; not all cells managed to reposition the laco regions ( finlan et al . , 2008 ; kumaran and spector , 2008 ; reddy et al . , 2008 ) . moreover , many transcriptional regulators dissociate from chromatin during mitosis and reassociate thereafter , and it is reasonable to presume that the stoichiometry and binding affinities of transcription factors present at the tdp would have a profound influence on the resulting chromatin architecture ( egli et al . , 2008 ) . as chromatin decondenses , it likely experiences a series of stochastic inter- and intra - chromosomal interactions of variable stabilities , facilitated by the high mobility of chromatin at this time ( chubb et al . , 2002 ; this would create the opportunity to selectively reinforce or discourage certain interactions to reinstate or alter chromatin folding or positioning , which has been proposed to maintain active and repressed states of gene expression ( fraser and bickmore , 2007 ; deng and blobel , 2010 ) . this model implies that the architecture assembled at the tdp affects the time and thus type of chromatin assembly during the upcoming s phase , which in turn will influence the probability of reassembling the same chromatin architecture in the next cell cycle . this could create a self - reinforcing feedback loop that would tend toward maintaining a 3d architecture characteristic of the previous cell cycle but that would be vulnerable to change at each tdp . early- and late - replicating chromatin domains are labeled as red or green , respectively , with light colors representing lack of rtds and bright colors representing the presence of rtds . because replication timing and spatial organization of chromatin are established simultaneously during early g1 phase at the tdp ( dimitrova and gilbert , 1999 ) and because there is a strong genome - wide correlation between 3d chromosome architecture and replication timing ( ryba et al . , 2010 ) , it is hypothesized that spatial reorganization at the tdp drives the assembly of rtds , potentially by creating subnuclear compartments that set thresholds for initiation of replication ( gilbert , 2001 ) . these rtds are maintained until the time of replication in s phase . during replication , the potential rtds are modified or removed at the replication fork , which is indicated by early - replicating domains changing to light colors first , followed by late - replicating domains . in g2 phase , there are no rtds on chromatin , but the general spatial organization is maintained until being disrupted during mitosis . if cells withdraw from the cell cycle and enter quiescence , aspects of the spatial organization of chromatin change but rtds remain intact , and upon return to the cell cycle , replication proceeds in the normal temporal order despite spatial disruption . as developed in the text , dismantling and reassembling higher - order chromosome architecture may provide a window of opportunity to reprogram 3d architecture and replication timing to influence cellular identity in response to extracellular cues during differentiation . once established , replication timing influences the type of chromatin assembled ( lande - diner et al . , 2009 ) , invoking a domain - wide change in chromatin structure that in turn may serve to reinforce maintenance of the new 3d architecture at each tdp . given that this window of time is small ( the first hours after mitosis ) and that remodeling chromatin architecture is probabilistic , this model provides an explanation for the inefficiency of cellular reprogramming in ipsc technology , the ability of cell division to improve reprogramming efficiency , and the ability of cell synchronization to improve animal cloning ( egli et al . , 2008 ) . in addition , the finding that quiescent cells retain rtds could help to explain how an adult stem cell , emerging from quiescence after stimulation by specific differentiation factors , can ensure self - renewal and maintenance of the stem cell population before committing to differentiation . such a stem cell would retain rtds regardless of the stimuli and assemble chromatin in the temporal order dictated by the prior tdp . this would preserve a memory of the stem cell identity until mitosis , when cell division could renew the stem cell . if the emerging cell was to commit before cell division , it would abrogate the possibility of self - renewal . another interesting nuance of this model relates to the fact that the length of g1 phase is very short in mouse escs ( panning and gilbert , 2005 ) . in addition , chromatin is very dispersed relative to differentiated cells ( hiratani et al . , 2010 ) , and there are a larger number of smaller early- and late - replicating domains ( hiratani et al . , 2008 ) . it is possible that under these conditions , replication initiates so rapidly after mitosis that there is little time to reorganize the nucleus , potentially contributing to the plasticity of pluripotent cells ( hiratani et al . , 2008 ) . the length of g1 phase in human escs is still a matter of debate ( white and dalton , 2005 ; neganova and lako , 2008 ) but is likely to be similar to mouse escs and should be resolved once the pluripotent states of human escs grown under different conditions are clarified ( hanna et al . , 2010 ) . indeed , lengthening g1 phase appears to be central to at least some differentiation pathways ( zhang et al . first , in cycling stem cells , if one stimulates differentiation , individual cells that are exiting mitosis should differentiate more rapidly than cells at other times during the cell cycle . second , adult stem cells emerging from quiescence should require mitosis to initiate the process of differentiation , and this window should close again at the following tdp . practically speaking , this second prediction is similar to the first , but the implications are that quiescent cells are not poised or vulnerable to certain differentiation signals despite their altered chromatin organization . moreover , this second prediction contradicts as yet unsubstantiated claims that nuclei from quiescent cells are more efficient substrates for animal cloning ( kasinathan et al . , 2001 ) . sophisticated live cell imaging systems that can track individual cells throughout multiple cell cycles , coupled with fluorescent beacons as indicators of cell fate transitions , should allow these predictions to be tested . if the tdp represents a critical time window for reprogramming , then the window should not close until several hours after nuclear reassembly and the resumption of transcription , when the completion of chromatin repositioning is observed . if , instead , mitosis is a more critical time period ( e.g. , through the eviction of transcription factors [ egli et al . , 2008 ] or binding of factors specifically in mitosis [ john and workman , 1998 ; zaidi et al . , 2010 ] ) , the window should close considerably earlier . similarly , these same experiments would also be able to identify whether another phase of the cell cycle , such as s phase or g2 phase ( during which chromatin mobility is restrained ) , is a more critical window of plasticity or whether cells are equally vulnerable to reprogramming regardless of their position in the cell cycle , either of which would also rule out the proposed hypothesis . of course , the markers chosen to assess differentiation will be critical because many genes are likely to be regulated by mechanisms that are independent of 3d architecture . in fact , we have found that transcription from cpg - rich promoters is independent of replication timing changes , implying that regulation of many genes is independent of higher - order chromatin folding ( hiratani et al . , 2008 ; hiratani et al . , 2010 ) . in conclusion , spatial compartmentalization of chromatin is a cell cycle regulated feature of chromosome organization that may dictate the time and place of chromatin assembly in the upcoming s phase , which would implicate the tdp as an important regulatory decision point for cellular identity . therefore , more thought , discussion , and experimentation into the role that events occurring at the tdp might play in maintaining stable cell identities during development and disease are needed .
recent findings suggest that large - scale remodeling of three dimensional ( 3d ) chromatin architecture occurs during a brief period in early g1 phase termed the replication timing decision point ( tdp ) . in this speculative article , i suggest that the tdp may represent an as yet unappreciated window of opportunity for extracellular cues to influence 3d architecture during stem cell fate decisions . i also describe several testable predictions of this hypothesis .
You are an expert at summarizing long articles. Proceed to summarize the following text: a 33-year - old right - handed male took a pneumatic nail gun and shot himself 10 times in the head : 5 times on the right , and 5 on the left . he reportedly suffered no obvious immediate sequelae : he was found well and watching television by his parents who compelled him to attend an emergency room when he informed them what he had done . he was subsequently transferred to our tertiary care hospital for definitive neurosurgical management . on the way , he had a focal seizure . on arrival , he was immediately assessed by the neurosurgical service where his initial neurological examination revealed that he was oriented in all 3 spheres , with a glasgow coma scale score of 15 . his pupils were equal and reactive to light bilaterally , and he had full visual fields . in addition , a left facial droop , left tongue deviation , right uvula deviation , mild dysarthria , subtle left sided motor weakness , and questionable decreased sensation over the left side were also noted . due to the history of a focal seizure , he was loaded with 1 g of phenytoin iv , and treated subsequently with a 7-day course of the anti - epileptic medication . the initial skull x - ray and non - contrast enhanced ct imaging revealed 10 metallic foreign bodies with a nail appearance projected over the temporal and occipitoparietal area bilaterally ( figures 1 & 2 ) . a decision was made by the neurosurgical service to bring him to the surgical suite on an emergent basis where he underwent bilateral small craniectomies and craniotomies for removal of each of the foreign bodies . post - operative ct imaging revealed only small multiple foci of intraparenchymal hemorrhage and associated subarachnoid hemorrhage . in addition , ct angiogram carried out on post - operative day 2 did not reveal any post - traumatic pseudoaneurysms or vasospasm . a repeat non - contrast ct scan demonstrated complete resolution of all of the intracranial hemorrhages . a 33-year - old right - handed male took a pneumatic nail gun and shot himself 10 times in the head : 5 times on the right , and 5 on the left . he reportedly suffered no obvious immediate sequelae : he was found well and watching television by his parents who compelled him to attend an emergency room when he informed them what he had done . he was subsequently transferred to our tertiary care hospital for definitive neurosurgical management . on the way , he had a focal seizure . on arrival , he was immediately assessed by the neurosurgical service where his initial neurological examination revealed that he was oriented in all 3 spheres , with a glasgow coma scale score of 15 . his pupils were equal and reactive to light bilaterally , and he had full visual fields . in addition , a left facial droop , left tongue deviation , right uvula deviation , mild dysarthria , subtle left sided motor weakness , and questionable decreased sensation over the left side were also noted . due to the history of a focal seizure , he was loaded with 1 g of phenytoin iv , and treated subsequently with a 7-day course of the anti - epileptic medication . the initial skull x - ray and non - contrast enhanced ct imaging revealed 10 metallic foreign bodies with a nail appearance projected over the temporal and occipitoparietal area bilaterally ( figures 1 & 2 ) . a decision was made by the neurosurgical service to bring him to the surgical suite on an emergent basis where he underwent bilateral small craniectomies and craniotomies for removal of each of the foreign bodies . post - operative ct imaging revealed only small multiple foci of intraparenchymal hemorrhage and associated subarachnoid hemorrhage . in addition , ct angiogram carried out on post - operative day 2 did not reveal any post - traumatic pseudoaneurysms or vasospasm . a repeat non - contrast ct scan demonstrated complete resolution of all of the intracranial hemorrhages . with the growing use of pneumatic nail guns both in the construction industry and by home consumers , penetrating craniofacial injuries caused by these devices are on the rise . most of these injuries are primarily work related accident,2 - 4 but they are also well - reported as intentionally self - inflicted.5 preoperative evaluation should always include thin cut ct scans allowing for multiplanar reconstruction . in addition , assessment of the cerebral vasculature is prudent especially if the nail appears to traverse a major vessel although the incidence of vascular injury secondary to nail gun injury is low . trauma to the cerebrovascular system has been associated with a wide variety of pathologic consequences including : arterial dissection , pseudoaneurysm , arteriovenous fistula , arterial or venous rupture or thrombosis,6 vessel perforations , carotid - cavernous fistula , and even fatal epistaxis.2 while catheter angiography has been the primary radiographic modality for diagnosing the extent of cerebrovascular trauma,7 ct angiography is an attractive screening imaging modality owing to the ease and rapidity of obtaining these images in the trauma setting . however , its sensitivity due to scatter artifact from the metallic foreign bodies is unclear.8 nonetheless plain ct scan as well as ct angiograms are well - reported initial imaging modalities before surgical removal.7,9 most of these injuries usually have relatively favorable neurological outcomes as long as the major cerebral vasculature and brainstem are not disrupted by the path of the penetrating nail.10 the primary and most commonly described vascular injury is the formation of a traumatic intracranial pseudoaneurysm , which necessitates both preoperative and follow - up cerebral angiography.8 traumatic intracranial aneurysms are rare and they tend to manifest clinically within 3 weeks after the time of injury , associated with a 50% mortality rate.11 because of the delayed visualization of traumatic pseudoaneurysms and the high risk of their formation in the setting of a nail gun injury , routine angiography should be performed in all patients between one to 2 weeks post injury.11 infectious complications of penetrating head trauma are high , and include local wound infection , the formation of cerebral abscess , ventriculitis , and meningitis . however , the use of broad spectrum antibiotic prophylaxis is only recommended in penetrating neurotrauma although without established guidelines.12 in our patient , after discussion with our infectious disease colleagues , the patient received broad spectrum antibiotics for 5 days . surgical treatment should be performed within 12 hours of the injury to decrease the risk of infectious complications.13 however , the technique to use is still debatable . no statistically significant advantage of one technique over the other has been described in reports of morbidity and mortality rates associated between a craniotomy or craniectomy.14 whichever technique used , the mainstay of these injuries is the treatment goal of removal of the foreign object whether by removal under local anesthetics with the patient awake or emergent craniotomy under general anesthesia . many single nail gun injuries were removed under local anesthetics with the patient awake.8 blind removal is usually uneventful , however there have been complications arising such as subdural hematomas and even intraparenchymal hemorrhage . this has lead to the suggestion that all penetrating foreign bodies must be removed under direct vision.15 while most would proceed with a standard craniotomy around the foreign object , spennato and colleagues16 describe the use of a double concentric craniotomy allowing for removal under direct visualization , without transmitting any undue forces to the underlying structures . when blind removal without formal craniotomy is judged appropriate , the availability of locking pliers ( of the vise grip style ) can be extremely helpful as their strong grip even with purchase limited to the edges of the nail head can permit extraction despite the usual firm implantation in the bone . the use of general anesthetic during removal is recommended for patients with more than 2 nails , high risks areas , or those in whom vascular injury is evident on preoperative imaging . should there be evidence of injuries involving vascular structures , preoperative therapeutic embolisation should be considered . this allows angiographic visualization with endovascular control of the proximal injured vessel if needed , and immediate follow - up angiography post - removal . in addition , this is a feasible sole treatment technique where surgical management promises to be technically difficult or poorly tolerated by the patient , or when poor neurological condition does not warrant craniotomy . many patients presenting with intracranial injuries from nail gun misuse have an associated psychiatric disturbance , usually schizophrenia or major depressive disorder.17 as such , not only is it important to involve other surgical specialties for combined intracranial and extracranial injury , successful treatment requires the additional collaborative expertise of colleagues from psychiatry . in conclusion , while most intracranial penetration from the use of a nail gun is single entry , here we present a case with 10 penetrating nail gun injuries . a careful clinical and radiologic examination is needed followed by extraction of the foreign body . most patients survive with good neurological outcome despite the dramatic appearance of the radiographic images .
penetrating craniocerebral injuries from nail gun use are rare . we describe a case of 10 self - inflicted nail gun injuries with intracranial penetrations . we also review the literature and discuss management strategies of such craniocerebral trauma . a 33-year - old male with a long - standing history of severe depression took a nail gun and sustained 10 penetrating intracranial injuries . initial neuroimaging revealed 10 penetrating nails , all sparing the major cerebral vasculature . immediate surgical removal was undertaken in the surgical suite using a combination of craniotomies , craniectomies , and blind removal . intracranial injuries from self - inflicted nail gun misuse is becoming increasingly more frequent . initial appropriate clinical decision - making are critical in preventing further cortical or vascular damage .
You are an expert at summarizing long articles. Proceed to summarize the following text: mucopolysaccharidosis vi ( mps vi ; omim # 253200 , maroteaux - lamy syndrome ) is an autosomal recessive disorder caused by lysosomal enzyme n - acetylgalactosamine 4-sulfatase ( arylsulfatase b , or asb ) deficiency with a clinical spectrum of mild to severe phenotypes . decreased asb enzyme activity leads to impaired degradation of the glycosaminoglycan ( gag ) dermatan sulfate . the resulting gag accumulation in the lysosomes of connective tissue causes a chronic progressive disorder characterized by significant functional impairment and shortened lifespan . patients with rapidly progressing disease often have coarse face , short stature , joint and skeletal deformities , corneal clouding , recurrent respiratory and ear infections , compromised cardiovascular and pulmonary function , spinal cord compression , and ultimately become wheelchair - bound or bedridden with early mortality in the late teens to early twenties . although patients with mps vi typically do not have neurocognitive impairment , their learning and development could be affected by physical limitations , particularly with vision deficit and hearing loss , . mps vi is an ultra - rare genetic disorder with a reported incidence of 0.140.38/100,000 live births . previous human clinical trials have demonstrated that enzyme replacement therapy ( ert ) for mps vi with recombinant human n - acetylgalactosamine 4-sulfatase ( rhasb ; galsulfase ; naglazyme ) significantly improves physical endurance , reduces urinary gag , and has an acceptable safety profile , , , , . giugliani et al . conducted a resurvey study of mps vi patients to obtain 10-year follow - up data that included medical histories and clinical assessments ( n = 59 ) , and survival status over 12 years ( n = 117 ) . they reported that long - term ert resulted in improvements in pulmonary function and endurance , stabilized cardiac function and increased survival . however , information on the long - term effects of ert in asian mps vi patients is limited . in this study , we report our findings on the long - term effects of galsulfase treatment in 9 taiwanese mps vi patients . data from 9 patients with mps vi ( 4 males and 5 females ) who received , or are currently receiving , ert with galsulfase ( 1.0 mg / kg / week intravenously ) between march 2004 and december 2015 in 5 medical centers in taiwan , were retrospectively reviewed . medical institutions included mackay memorial hospital , china medical university hospital , national cheng kung university hospital , changhua christian hospital , and national taiwan university hospital . the patients ' ages at which treatment was begun ranged widely from 1.4 to 21.1 years , and the duration of therapy ranged from 6.2 to 11.2 years . we routinely assessed a set of biochemical and clinical responses each year during treatment . written informed consent for ert was obtained from a parent for children and from patients over 18 years . the study was approved by the ethics committee of mackay memorial hospital , taipei , taiwan . all patients had clinical manifestations of mps vi , and diagnosis was confirmed by two - dimensional electrophoresis of urinary gags and asb enzyme assay in serum , leukocytes and/or fibroblasts . height and weight were transformed to standard deviation scores ( z scores ) on the basis of a standard growth table for the taiwanese population . prior to each intravenous infusion , patients were pre - medicated with diphenhydramine ( 0.5 mg / kg body weight ) . galsulfase was diluted in 0.9% saline and administered at 1.0 mg / kg over 4 h once weekly according to product label administration instructions . urinary gags and white blood cell asb levels were measured at baseline and every year during treatment . clinical functional assessments varied somewhat by institution and may have included a 6-minute walk test ( 6mwt ) , 3-minute stair climb test ( 3msct ) , joint range of motion , coin picking - up test , spirometry , echocardiography , abdominal ultrasonography , bone mineral density , and hearing test . range of motion of the shoulders was measured with a goniometer by physical therapists . the goniometer assessments included both active and passive shoulder flexion , extension , and lateral rotation , and were consistent at all centers . forced vital capacity ( fvc ) and forced expiratory volume in 1 s ( fev1 ) were evaluated by standard spirometry techniques according to american thoracic society guidelines , . the cardiac ultrasound systems used were philips sonos 5500/7500 system ( andover , ma , usa ) , equipped with electronic transducers from 2 to 8 mhz . diastolic and systolic diameters were measured using the m - mode , and the systolic function of the left ventricle was evaluated through the ejection fraction obtained by the teichholz method [ v = 7d / ( 2.4 + d ) , where v = lv volume and d = lv diameter ] . diastolic filling was established using the e / a ratio by measuring mitral - inflow as determined by pattern - peak early filling ( e ) and late filling ( a ) velocities . a reversed e / a ratio ( e / a ratio < 1 ) was considered diastolic dysfunction . severity of valvular stenosis and regurgitation were estimated and graded on the following scores : 0 ( none ) , 1 ( mild ) , 2 ( moderate ) , and 3 ( severe ) based on the european society of cardiology / american society of echocardiography guidelines , . the data of left ventricular mass index ( lvmi ) , the thicknesses of the interventricular septum diameter in diastole ( ivsd ) , and the thicknesses of the left ventricular posterior wall diameter in diastole ( lvpwd ) obtained by serial echocardiographic assessments were recorded . these values were compared with normal values according to the study of kampmann et al . . left ventricular mass ( lvm ) was calculated according to the american society of echocardiography simplified cubed equation . lvm was indexed ( lvmi ) by height to normalize heart size to body size . the lvmi was also calculated using the devereux formula and indexed by body surface area with normal values according to the report of poutanen et al . . all above echocardiographic values were transformed into a z score derived by subtracting the mean reference value from an individual observed value , and then dividing the difference by the standard deviation from the reference value . abdominal ultrasonographic examinations were performed using high - resolution b - mode ultrasonography ( sa-700a , toshiba , tokyo , japan ) with a 3.5-mhz curved array transducer . liver size and spleen size were measured in comparison with the normal reference values for different body height of children . the absolute value of bone mineral density ( bmd ) and height - for - age ( haz ) adjusted bmd z scores were evaluated by dual energy x - ray absorptiometry ( dxa ) as previously described . the assessment of hearing loss by pure - tone audiometry ( pta ) was performed as previously described with the collection of the values of air conduction ( ac ) , bone conduction ( bc ) , and air - bone gap . joint pain and stiffness questionnaire scores ( disability index ) were assessed by an analog scale based on the health assessment questionnaire ( haq ) , for patients who were > 18 years of age or childhood health assessment questionnaire ( chaq ) completed by a parent or caregiver for younger patients . in 3 cases a manual dexterity test was performed where the patient was asked to pick up 10 coins and place them in a cup while their elbow was allowed to rest on the table ; the time required to complete the task was recorded . all 9 patients were assessed using the disability index , with an overall score that addresses eight categories of activity , including dressing and grooming , arising , eating , walking , hygiene , reach , grip , and common daily activities . the latest results of examinations for these 9 patients receiving ert were compared with baseline data . descriptive statistics were calculated , including means , standard deviations , and percentage change over time . data from 9 patients with mps vi ( 4 males and 5 females ) who received , or are currently receiving , ert with galsulfase ( 1.0 mg / kg / week intravenously ) between march 2004 and december 2015 in 5 medical centers in taiwan , were retrospectively reviewed . medical institutions included mackay memorial hospital , china medical university hospital , national cheng kung university hospital , changhua christian hospital , and national taiwan university hospital . the patients ' ages at which treatment was begun ranged widely from 1.4 to 21.1 years , and the duration of therapy ranged from 6.2 to 11.2 years . we routinely assessed a set of biochemical and clinical responses each year during treatment . written informed consent for ert was obtained from a parent for children and from patients over 18 years . the study was approved by the ethics committee of mackay memorial hospital , taipei , taiwan . all patients had clinical manifestations of mps vi , and diagnosis was confirmed by two - dimensional electrophoresis of urinary gags and asb enzyme assay in serum , leukocytes and/or fibroblasts . height and weight were transformed to standard deviation scores ( z scores ) on the basis of a standard growth table for the taiwanese population . prior to each intravenous infusion , patients were pre - medicated with diphenhydramine ( 0.5 mg / kg body weight ) . galsulfase was diluted in 0.9% saline and administered at 1.0 mg / kg over 4 h once weekly according to product label administration instructions . urinary gags and white blood cell asb levels were measured at baseline and every year during treatment . clinical functional assessments varied somewhat by institution and may have included a 6-minute walk test ( 6mwt ) , 3-minute stair climb test ( 3msct ) , joint range of motion , coin picking - up test , spirometry , echocardiography , abdominal ultrasonography , bone mineral density , and hearing test . range of motion of the shoulders was measured with a goniometer by physical therapists . the goniometer assessments included both active and passive shoulder flexion , extension , and lateral rotation , and were consistent at all centers . forced vital capacity ( fvc ) and forced expiratory volume in 1 s ( fev1 ) were evaluated by standard spirometry techniques according to american thoracic society guidelines , . the cardiac ultrasound systems used were philips sonos 5500/7500 system ( andover , ma , usa ) , equipped with electronic transducers from 2 to 8 mhz . diastolic and systolic diameters were measured using the m - mode , and the systolic function of the left ventricle was evaluated through the ejection fraction obtained by the teichholz method [ v = 7d / ( 2.4 + d ) , where v = lv volume and d = lv diameter ] . diastolic filling was established using the e / a ratio by measuring mitral - inflow as determined by pattern - peak early filling ( e ) and late filling ( a ) velocities . a reversed e / a ratio ( e / a ratio < 1 ) was considered diastolic dysfunction . severity of valvular stenosis and regurgitation were estimated and graded on the following scores : 0 ( none ) , 1 ( mild ) , 2 ( moderate ) , and 3 ( severe ) based on the european society of cardiology / american society of echocardiography guidelines , . the data of left ventricular mass index ( lvmi ) , the thicknesses of the interventricular septum diameter in diastole ( ivsd ) , and the thicknesses of the left ventricular posterior wall diameter in diastole ( lvpwd ) obtained by serial echocardiographic assessments were recorded . these values were compared with normal values according to the study of kampmann et al . . left ventricular mass ( lvm ) was calculated according to the american society of echocardiography simplified cubed equation . lvm was indexed ( lvmi ) by height to normalize heart size to body size . the lvmi was also calculated using the devereux formula and indexed by body surface area with normal values according to the report of poutanen et al . . all above echocardiographic values were transformed into a z score derived by subtracting the mean reference value from an individual observed value , and then dividing the difference by the standard deviation from the reference value . abdominal ultrasonographic examinations were performed using high - resolution b - mode ultrasonography ( sa-700a , toshiba , tokyo , japan ) with a 3.5-mhz curved array transducer . liver size and spleen size were measured in comparison with the normal reference values for different body height of children . the absolute value of bone mineral density ( bmd ) and height - for - age ( haz ) adjusted bmd z scores were evaluated by dual energy x - ray absorptiometry ( dxa ) as previously described . the assessment of hearing loss by pure - tone audiometry ( pta ) was performed as previously described with the collection of the values of air conduction ( ac ) , bone conduction ( bc ) , and air - bone gap . joint pain and stiffness questionnaire scores ( disability index ) were assessed by an analog scale based on the health assessment questionnaire ( haq ) , for patients who were > 18 years of age or childhood health assessment questionnaire ( chaq ) completed by a parent or caregiver for younger patients . in 3 cases a manual dexterity test was performed where the patient was asked to pick up 10 coins and place them in a cup while their elbow was allowed to rest on the table ; the time required to complete the task was recorded . all 9 patients were assessed using the disability index , with an overall score that addresses eight categories of activity , including dressing and grooming , arising , eating , walking , hygiene , reach , grip , and common daily activities . the latest results of examinations for these 9 patients receiving ert were compared with baseline data . descriptive statistics were calculated , including means , standard deviations , and percentage change over time . the diagnosis of mps vi was confirmed in all 9 patients according to urinary gag profiles , asb enzyme activity level , and dna mutation analysis . the z scores for height and weight at baseline were 5.4 3.4 and 1.7 1.3 , respectively ( table 1 ) . the baseline clinical assessment data was described as our previous study for these 9 taiwanese patients with mps vi in 2010 . after 6.2 to 11.2 years of treatment , there was a 69% decrease in urinary excretion of gag , indicating biomarker improvement ( table 1 , table 2 , fig . measures of function also improved , including 6mwt [ in 6 patients , mean 150-meter ( 59% ) increase over baseline ] and 3msct [ in 3 patients , mean 60-step ( 46% ) increase ] . the time requirement of picking up 10 coins and putting them into a cup in 3 patients decreased 15 s ( 33% ) . shoulder range of motion improved in all 9 patients , as did the disability index ( 0.42 points , 21% ) . the 4 patients who underwent pulmonary function testing had improved fev1 by 0.29 l ( 59% ) and fvc by 0.42 l ( 80% ) ( table 1 , table 2 ) . the 5 patients who underwent echocardiographic evaluations had positive effects on cardiac - wall diameters . after ert , the mean lvmi z score decreased from 3.18 to 1.88 , the mean ivsd z score decreased from 6.86 to 2.33 , and the mean lvpwd z score decreased from 1.70 to 0.30 ( fig . four of these 5 patients had improved cardiac diastolic function according to the improvement of e / a ratio ( fig . however , the severity of valvular stenosis or regurgitation remained stable and did not show improvement despite ert ( fig . s3 ) . liver and spleen size decreased or remained unchanged after ert in all 9 patients by abdominal ultrasonographic assessments ( table 3 ) . three patients who underwent baseline and follow - up dxa screening after receiving ert for 3.8 to 7.4 years showed an increase in absolute bmd values , as well as an improvement in haz adjusted bmd z score ( fig . three patients who had follow - up pta after undergoing ventilation tube insertion while receiving ert for 5.2 to 8.5 years showed improvements in ac and bc of the better ear , as well as a decrease in the air - bone gap ( fig . patient 4 did grow somewhat in height , but none of the patients achieved the third percentile for height ( fig . three patients had hypersensitivity reactions ( skin itching / urticaria , dyspnea , fever ) at some point during their treatment . however , following pre - medication with oral antihistamines , steroids and antipyretics , all were able to tolerate ert despite mild reactions continuing for about 5 months in 2 of the 3 individuals . with continued pre - medication , three patients had hypersensitivity reactions ( skin itching / urticaria , dyspnea , fever ) at some point during their treatment . however , following pre - medication with oral antihistamines , steroids and antipyretics , all were able to tolerate ert despite mild reactions continuing for about 5 months in 2 of the 3 individuals . with continued pre - medication , this is the first report to demonstrate clinical benefits of long - term ert in taiwanese patients with mps vi . as far as we are aware , the 9 cases presented here are the only mps vi patients in taiwan who are currently receiving ert . our review clearly demonstrates both biochemical and clinical functional improvement in all 9 patients after 611 years of ert . although several studies have shown that short - term ert for mps vi patients positively affects joint mobility , liver and spleen size , cardiac parameters , pulmonary function , and urinary gags levels , , , , , , , there is very limited literature reporting the long - term ert effects for patients with mps vi . swiedler et al . carried out a cross - sectional survey of 121 patients with mps vi , and reported an association between high urinary gag values ( > 200 g / mg creatinine ) and a worse clinical course of disease although the clinical manifestation varied widely among patients . in our study , all individuals had urinary gag values above 200 g / mg creatinine at baseline suggesting the severe phenotype may be more prevalent in the taiwanese population ( table 1 ) . the observed decrease in urinary gag excretion seen with long - term ert paralleled improvements in patients ' functional status . our results thus support the effectiveness of ert for mps vi , with functional improvement accompanying biochemical response . however , some aspects of disease phenotype are unaffected by long - term ert . for example , no improvements in skeletal dysplasia or corneal clouding were observed in galsulfase treated taiwanese patients , which were in accordance with those of mcgill et al . . reported that fev1 and fvc increased over baseline by 11% and 17% , respectively , after 96 weeks of ert for 56 patients with mps vi . this improvement in pulmonary function is one factor underlying the increased physical endurance documented in the 6-minute and 12-minute walk tests . ert appeared to be effective in reducing cardiac hypertrophy in our 5 patients who had detailed echocardiographic examinations . four of these 5 patients had improved cardiac diastolic function according to the improvement of e / a ratio . these results suggest that ert may have some effect on gag accumulation in cardiac tissue , and thus was effective in reducing cardiac hypertrophy and diastolic dysfunction . our results are consistent with other studies describing the cardiac effects of ert for patients with mps vi , , . the older patients in our study had hepatosplenomegaly , while the younger patients showed relatively normal liver and spleen size ( table 3 ) , reflecting the progressive nature of the disease . reported that after ert for 1.3 to 5.4 years , liver and spleen size decreased in all eleven mps vi patients when assessed manually by physicians . similarly , our results showed that liver and spleen size decreased or remained unchanged after ert for all 9 patients as measured by abdominal ultrasonography . there is a paucity of literature describing the effects of ert on bmd in mps , . in our study , 3 mps vi patients who underwent follow - up dxa after receiving ert for 3.8 to 7.4 years showed an increase in absolute bmd values , as well as an improvement in their haz adjusted bmd z scores . improvements in bmd in mps vi patients receiving ert may occur through multiple mechanisms including reduced gag storage in the bones , increased muscle strength and endurance , and improved pulmonary function and mobility , , , . described that 2 of 3 patients with mps vi improved conductive hearing loss after ert , possibly due to ert - related decrease in occurrence of upper airway infections . in our study , 3 mps vi patients improved or stabilized their bc values [ sensorineural hearing loss component ] after receiving ert for 5.2 to 8.5 years . improvement of conductive hearing loss in mps patients receiving ert may be due to fewer upper airway infections , as well as reducing middle ear effusions causing pressure on the eustachian tube . however , it remains unclear whether sensorineural hearing loss has a congenital basis or is acquired secondarily to deposition of gags in the inner ear or central nervous system . the issue concerning the effect of ert for mps on sensorineural hearing loss may be resolved after longer follow - up . overall , our experience with treating taiwanese patients is thus consistent with the results of ert elsewhere in the world . in our series , none of the patients reached the third percentile of the height curve , suggesting a limited effect of ert on growth ( fig . are similar . in cats treated from birth , ert prevented or slowed skeletal dysplasia , however , whether it is possible in humans to identify the disease early enough to begin ert before irreversible skeletal pathology has occurred remains to be seen . our results showed that 7 out of 9 patients had spinal cord compression symptoms , which were consistent with those of scarpa et al . . adverse events , such as skin rash , dyspnea , and pyrexia , were reported in clinical trials of galsulfase for mps vi , , , . a third of our patients had similar symptoms , but the reactions were easily managed . none of the 3 had serious sequelae , and all 3 were able to continue with treatment . this retrospective and multicenter study did not compare the effects of ert in these patients with untreated control subjects . meanwhile , clinical assessments varied by institutions and treating physicians . besides , the range of age at which treatment started was quite wide , as was the degree of disease severity . however , our experience reflects what clinicians probably come across in daily practice with unselected patients . this retrospective and multicenter study did not compare the effects of ert in these patients with untreated control subjects . meanwhile , clinical assessments varied by institutions and treating physicians . besides , the range of age at which treatment started was quite wide , as was the degree of disease severity . however , our experience reflects what clinicians probably come across in daily practice with unselected patients . in our case series of 9 taiwanese patients with mps vi , long - term ert with galsulfase improved endurance , mobility , joint function , pulmonary function , cardiac hypertrophy and diastolic dysfunction , liver size and spleen size , while reducing urinary gag excretion . ert was well - tolerated , with mild hypersensitivity reactions in 3 patients easily managed with routine pre - medication therapy . ert for treatment of mps vi has been endorsed by the national health insurance program in taiwan since february 2006 . our clinical experience confirms that long - term ert is clearly beneficial for taiwanese patients with mps vi , just as it is for other global mps vi populations . hyl performed acquisition , statistical analysis and interpretation of data , and drafting of the manuscript . spl participated in design of the study , interpretation of the data and helped to draft the manuscript . chw , yhc , ymw , fjt , yyc , sjl , hpp , dmn , wlh and yyk were responsible for patient screening . fig . s1parameter changes of echocardiographic assessments at baseline and after 5.28.4 years of enzyme replacement therapy ( ert ) for 5 patients with mucopolysaccharidosis vi ( patients no lvmi , left ventricular mass index ; ivsd , the thicknesses of the interventricular septum diameter in diastole ; lvpwd , the thicknesses of the left ventricular posterior wall diameter in diastole . s2 e / a ratio changes of echocardiographic assessments of diastolic dysfunction at baseline and after 5.910.8 years of enzyme replacement therapy ( ert ) for 5 patients with mucopolysaccharidosis vi ( patients no . 1 to 5 ) . e / a ratio , ratio of the early ( e ) to late ( a ) ventricular filling velocities . e / a ratio 1 , normal diastolic function ; e / a ratio < 1 , impaired relaxation . fig . s3 parameter changes of echocardiographic assessments of valvular heart diseases at baseline and after 5.910.8 years of enzyme replacement therapy ( ert ) for 5 patients with mucopolysaccharidosis vi ( patients no ms , mitral stenosis ; mr , mitral regurgitation ; as , aortic stenosis ; ar , aortic regurgitation . severity of valvular stenosis and regurgitation were estimated and graded on the following scores : 0 ( none ) , 1 ( mild ) , 2 ( moderate ) , and 3 ( severe ) . s4 baseline and follow - up absolute bone mineral density ( bmd ) values and height - for - age z score ( haz ) adjusted bmd z scores of 3 patients with mucopolysaccharidosis vi ( patients no . 1 to 3 ) baseline and follow - up pure - tone audiometry ( pta ) parameters of 3 patients with mucopolysaccharidosis vi ( patients no . 1 to 3 ) who underwent ventilation tube insertion and received enzyme replacement therapy ( ert ) for 5.2 to 8.5 years . s1 parameter changes of echocardiographic assessments at baseline and after 5.28.4 years of enzyme replacement therapy ( ert ) for 5 patients with mucopolysaccharidosis vi ( patients no lvmi , left ventricular mass index ; ivsd , the thicknesses of the interventricular septum diameter in diastole ; lvpwd , the thicknesses of the left ventricular posterior wall diameter in diastole . s2 e / a ratio changes of echocardiographic assessments of diastolic dysfunction at baseline and after 5.910.8 years of enzyme replacement therapy ( ert ) for 5 patients with mucopolysaccharidosis vi ( patients no . 1 to 5 ) . e / a ratio , ratio of the early ( e ) to late ( a ) ventricular filling velocities . e / a ratio 1 , normal diastolic function ; e / a ratio < 1 , impaired relaxation . fig . s3 parameter changes of echocardiographic assessments of valvular heart diseases at baseline and after 5.910.8 years of enzyme replacement therapy ( ert ) for 5 patients with mucopolysaccharidosis vi ( patients no ms , mitral stenosis ; mr , mitral regurgitation ; as , aortic stenosis ; ar , aortic regurgitation . severity of valvular stenosis and regurgitation were estimated and graded on the following scores : 0 ( none ) , 1 ( mild ) , 2 ( moderate ) , and 3 ( severe ) . s4 baseline and follow - up absolute bone mineral density ( bmd ) values and height - for - age z score ( haz ) adjusted bmd z scores of 3 patients with mucopolysaccharidosis vi ( patients no . 1 to 3 ) underwent enzyme replacement therapy ( ert ) for 3.8 to 7.4 years . baseline and follow - up pure - tone audiometry ( pta ) parameters of 3 patients with mucopolysaccharidosis vi ( patients no . 1 to 3 ) who underwent ventilation tube insertion and received enzyme replacement therapy ( ert ) for 5.2 to 8.5 years .
backgroundinformation regarding the long - term outcome of enzyme replacement therapy ( ert ) with recombinant human n - acetylgalactosamine 4-sulfatase ( rhasb , galsulfase , naglazyme , biomarin pharmaceutical inc . ) for taiwanese patients with mucopolysaccharidosis ( mps ) vi is limited.methodsnine taiwanese patients with mps vi ( 4 males and 5 females ; age range , 1.4 to 21.1 years ) treated with weekly intravenous infusions of galsulfase ( 1.0 mg / kg ) in 5 medical centers in taiwan were reviewed . a set of biochemical and clinical assessments were evaluated annually.resultsafter 6.2 to 11.2 years of galsulfase treatment , 6 patients experienced improvement over baseline in the 6-minute walk test by a mean of 150 m ( 59% change over time ) , and 3 patients also increased the 3-minute stair climb test by a mean of 60 steps ( 46% ) . in a manual dexterity test , 3 patients decreased the time required to pick up 10 coins and put the coins into a cup by 15 s ( 33% ) . shoulder range of motion in all 9 patients improved , and joint pain and stiffness questionnaire scores improved by 0.42 points ( 21% ) . four patients showed improved pulmonary function . five patients had positive effects on cardiac - wall diameters . four patients had improved cardiac diastolic function . liver and spleen sizes as measured by abdominal ultrasonography remained the same or decreased in all 9 patients . however , the severity degree of valvular stenosis or regurgitation did not show improvement despite ert . a mean overall 69% decrease in urinary glycosaminoglycan ( gag ) excretion indicated a satisfactory biomarker response.conclusionslong-term ert was beneficial and safe for taiwanese patients with mps vi . this treatment reduced urinary gag and had positive effects on a wide range of clinical functional assessments including endurance , mobility , joint function , pulmonary function , liver and spleen size , cardiac hypertrophy and diastolic dysfunction .
You are an expert at summarizing long articles. Proceed to summarize the following text: to describe a case in which descemet 's membrane interfered with aqueous humor drainage through an express mini shunt . a 70-year - old male , diagnosed with corticosteroid - induced glaucoma in his right eye , presented to our hospital . topical betamethasone treatment was discontinued , and the patient was treated with intravenous d - mannitol and acetazolamide , followed by oral acetazolamide , oral potassium l - aspartate , topical dorzolamide hydrochloride , topical carteolol hydrochloride , and topical latanoprost . however , his right intraocular pressure ( iop ) remained elevated . his postoperative iop was initially within the normal range , but it reincreased 1 month after surgery . we found that the descemet 's membrane was interfering with both the primary ( axial ) and reserve orifices at the tip of the express mini shunt . nd : yag laser membranotomy was performed and the patient 's iop again improved without any other medical treatment . descemet 's membrane interfered with aqueous humor drainage via express mini shunt , causing an increased iop , which was resolved by nd : yag laser membranotomy . . , usa ) mini glaucoma shunt a non - valved , mri - compatible , stainless steel device controls intraocular pressure ( iop ) by shunting aqueous humor from the anterior chamber to a subconjunctival reservoir , in a similar fashion as trabeculectomy . dahan and carmichael proposed implanting this device under a scleral flap to avoid complications associated with its placement under the conjunctiva , and other studies concluded that express implantation is most appropriate in patients with a high risk of complications , such as hypotony [ 2 , 3 ] . this case report involved a unique complication , which , to the best of our knowledge , has not been previously reported in relation to express implantation . in january 2014 , a 70-year - old male experienced painful vision loss in his right eye . several years earlier , he had been diagnosed with severe allergic conjunctivitis and had since then used topical betamethasone . following his vision loss , he was diagnosed with corticosteroid - induced glaucoma in his right eye , and his topical betamethasone use was terminated . he was treated with intravenous d - mannitol and acetazolamide , followed by oral acetazolamide , oral potassium l - aspartate , topical dorzolamide hydrochloride , topical carteolol hydrochloride , and topical latanoprost . after these treatments , the patient 's right iop was reduced to 2530 mm hg . the patient 's best - corrected visual acuity was 0.15 in his right eye and 1.2 in his left eye . goldmann perimetry showed advanced glaucomatous visual field loss in his right eye . due to the patient 's sustained elevated iop despite maximum medical therapy , it was decided to perform the insertion of an express glaucoma filtration device in his right eye in march 2014 . a subtenon dose of 1% preservative - free lidocaine was injected superonasally , followed by a fornix - based conjunctival dissection . a rectangular flap with a partial thickness of 3.5 3.5 mm was created , and sponges soaked in 0.4mg / ml mitomycin c were applied to the area for 3 min . the sponges were removed , and the area was irrigated with a balanced salt solution . after 2 2 mm deep sclerectomy , the anterior chamber was entered with a 26-gauge needle , inserted under the scleral flap in the center of the blue - gray transition zone , and an express drainage device ( model p-50 ) was introduced into the anterior chamber . the scleral flap was subsequently closed with 4 interrupted 100 nylon flap sutures , and the conjunctiva and tenon 's fascia were reapproximated to the limbus with 2 100 nylon compression sutures . the patient 's postoperative iop initially remained within the normal range , with a diffuse non - leaking bleb and a deep anterior chamber . however , at 1 month after the surgery , the iop again increased to 2530 mm hg . we found that both the primary ( axial ) and reserve orifices at the tip of the express mini shunt were impeded by descemet 's membrane ( fig . 1a ) . following the performance of the nd : yag laser membranotomy ( 1.6 mj , 14 shots ) ( fig . 1b ) , the patient 's iop improved to 1015 mm hg without any other medical treatment . in the case presently described , descemet 's membrane apparently interfered with aqueous humor inflow into an inserted express mini shunt , leading to a reincrease of the iop . nd : yag laser membranotomy promptly resolved this problem . during and immediately after surgery , we did not recognize any embolization of descemet 's membrane into the primary ( axial ) and reserve orifices at the tip of the express mini shunt . in the present case , the precise mechanism of this device - related complication remains unclear , but it is thought that the floating membrane fragment was yielded due to an error in the process of inserting the express mini shunt into the entry incision . first , we used a 26-gauge needle ( 0.455 mm ) for preincision , but the manufacturer recommends using a 25-gauge needle ( 0.405 mm ) to enter the anterior chamber . while this 0.05-mm difference may have promoted a tighter fixation of the device and less leakage of aqueous humor around the device , it could also be associated with a greater risk of peeling the descemet 's membrane off the cornea . second , the position of the pre - incision under the scleral flap was too close to the cornea and may have torn the descemet 's membrane . third , the direction of the express delivery system was too parallel to the descemet 's membrane . fourth , when the express mini shunt penetrated the anterior chamber , involuntary eyeball rotation may have occurred due to insufficient counterpressure applied to the opposite side of the eye or excessive resistance caused by the use of a 26-gauge needle for preincision . in conclusion , to avoid the presently described complication when setting an express mini shunt , one should be aware of the possibility of releasing a fragment of descemet 's membrane .
purposeto describe a case in which descemet 's membrane interfered with aqueous humor drainage through an express mini shunt . this problem was successfully solved by nd : yag laser membranotomy.case reporta 70-year - old male , diagnosed with corticosteroid - induced glaucoma in his right eye , presented to our hospital . topical betamethasone treatment was discontinued , and the patient was treated with intravenous d - mannitol and acetazolamide , followed by oral acetazolamide , oral potassium l - aspartate , topical dorzolamide hydrochloride , topical carteolol hydrochloride , and topical latanoprost . however , his right intraocular pressure ( iop ) remained elevated . we performed express shunt surgery in the patient 's right eye . his postoperative iop was initially within the normal range , but it reincreased 1 month after surgery . we found that the descemet 's membrane was interfering with both the primary ( axial ) and reserve orifices at the tip of the express mini shunt . nd : yag laser membranotomy was performed and the patient 's iop again improved without any other medical treatment.conclusiondescemet's membrane interfered with aqueous humor drainage via express mini shunt , causing an increased iop , which was resolved by nd : yag laser membranotomy .
You are an expert at summarizing long articles. Proceed to summarize the following text: radical prostatectomy ( rp ) is a challenging operation that demands high levels of surgical expertise and experience . rp can be performed with open or with minimally invasive laparoscopic or robot - assisted approaches . vesicourethral anastomosis is the most technically challenging part of the procedure and can be performed with either interrupted or running sutures , both in open rp and in laparoscopic prostatectomy . insufficient vesicourethral anastomosis can cause significant postoperative urinary extravasation , which can result in a longer catheterization time with patient discomfort and an increased risk of long - term anastomotic strictures . most urinary extravasation occurs immediately after surgery ; it is mostly self - limiting and resolves without additional intervention . however , further intervention is occasionally required for persistent vesicourethral anastomotic urinary extravasation , which if severe , can require reoperation and reanastomosis . thus far , only 2 previous studies have compared the use of interrupted anastomosis with running anastomosis for laparoscopic rp . in laparoscopic rp , running anastomosis is usually used , with reports that it is quicker and technically less challenging than interrupted anastomosis . the running anastomosis is theoretically more watertight and reinserting the catheter is safer and easier , because the catheter tip is less likely to pass posteriorly and extravesically . several studies have reported that running anastomosis is technically feasible and provides safe and early catheter removal in open rp . however , for open rp , most surgeons perform vesicourethral anastomosis by use of interrupted sutures because of the difficulty of anastomosis and the lack of studies comparing outcomes for open rp using running and interrupted techniques . here we describe the easier method of vesicourethral running sutures for open rp and compare perioperative outcomes of the two anastomosis approaches . after institutional review board approval of gangneung asan hospital was obtained , a retrospective review was carried out of the medical records of 112 patients who underwent open rp for prostate cancer performed by a single surgeon ( j.y.p . ) between 2006 and 2008 . patients who had hormone therapy or radiation therapy were excluded from the study , as were patients with missing 3-month follow - up data . open rp was performed up to the stage of vesicourethral anastomosis as per walsh 's technique . we used the technique described by walsh for interrupted anastomosis suturing . for the running anastomosis technique , we prepared a single anastomotic suture by tying the tails of two 3 - 0 monosyn ( monofilament ) sutures on a 5/8 circle needle . we used a modified version of the technique described by van velthoven et al . by using approximately 12 sutures ; initially , the knot was located at the 4 o'clock position of the bladder neck and anchored the anastomotic stitch at this location . one of the needles was sutured from the outside in to the left of the 4 o'clock position of the bladder neck ( fig . we proceeded with a clockwise running suture in the left lateral wall of the anastomosis from the 4 o'clock position of the bladder neck to the 8 o'clock position on the outside of the urethral wall . the needle was always driven full - thickness from the outside in into the bladder wall and from the inside out on the urethral stump . we used a nephrostomy catheter for facilitating in - to - out suture placement at the urethral stump instead of the urethral catheter or metal sound . the needle tip was placed to the inside of the nephrostomy catheter lumen ( fig . this makes it technically easier than using a metal sound or urethral catheter and prevents injury to the urethral stump ( fig . gentle traction was applied to the end of the suture to avoid loosening the suture and the other needle was driven from the outside in to the right of the 4 o'clock position of the bladder neck , and this suture was used to perform a counterclockwise running suture in the right bladder wall to the 2 o'clock position from the urethral stump ( fig . then , the left running suture was completed from the 8 o'clock to the 12 o'clock position and the right side was also completed from the 2 o'clock to the 12 o'clock position . after gentle traction of each suture , it is easy to access the site of the anastomosis . we then checked for vesicourethral anastomosis urine leaks after instillation of 150 ml of normal saline into the bladder . if patients were unable to void adequately , the urethral catheter was inserted for 3 to 5 additional days . cystography was repeated 5 to 7 days later to reevaluate the patient for catheter removal . all patients were followed up after open rp every 3 months for the first year , biannually during the second year , and annually thereafter . each visit included a clinical examination and prostate - specific antigen measurement . during follow - up continence was defined as no pads required for any activity that was routine before the operation . bladder neck contracture was detected by uroflowmetry and cystoscopy in patients complaining of voiding difficulty . clinicopathologic factors were compared between the 2 groups by using student t - test for continuous variables and pearson chi - square test for categorical variables . after institutional review board approval of gangneung asan hospital was obtained , a retrospective review was carried out of the medical records of 112 patients who underwent open rp for prostate cancer performed by a single surgeon ( j.y.p . ) between 2006 and 2008 . patients who had hormone therapy or radiation therapy were excluded from the study , as were patients with missing 3-month follow - up data . open rp was performed up to the stage of vesicourethral anastomosis as per walsh 's technique . we used the technique described by walsh for interrupted anastomosis suturing . for the running anastomosis technique , we prepared a single anastomotic suture by tying the tails of two 3 - 0 monosyn ( monofilament ) sutures on a 5/8 circle needle . we used a modified version of the technique described by van velthoven et al . by using approximately 12 sutures ; initially , the knot was located at the 4 o'clock position of the bladder neck and anchored the anastomotic stitch at this location . one of the needles was sutured from the outside in to the left of the 4 o'clock position of the bladder neck ( fig . we proceeded with a clockwise running suture in the left lateral wall of the anastomosis from the 4 o'clock position of the bladder neck to the 8 o'clock position on the outside of the urethral wall . the needle was always driven full - thickness from the outside in into the bladder wall and from the inside out on the urethral stump . we used a nephrostomy catheter for facilitating in - to - out suture placement at the urethral stump instead of the urethral catheter or metal sound . the needle tip was placed to the inside of the nephrostomy catheter lumen ( fig . this makes it technically easier than using a metal sound or urethral catheter and prevents injury to the urethral stump ( fig . gentle traction was applied to the end of the suture to avoid loosening the suture and the other needle was driven from the outside in to the right of the 4 o'clock position of the bladder neck , and this suture was used to perform a counterclockwise running suture in the right bladder wall to the 2 o'clock position from the urethral stump ( fig . then , the left running suture was completed from the 8 o'clock to the 12 o'clock position and the right side was also completed from the 2 o'clock to the 12 o'clock position . after gentle traction of each suture , it is easy to access the site of the anastomosis . we then checked for vesicourethral anastomosis urine leaks after instillation of 150 ml of normal saline into the bladder . if patients were unable to void adequately , the urethral catheter was inserted for 3 to 5 additional days . cystography was repeated 5 to 7 days later to reevaluate the patient for catheter removal . all patients were followed up after open rp every 3 months for the first year , biannually during the second year , and annually thereafter . each visit included a clinical examination and prostate - specific antigen measurement . during follow - up continence was defined as no pads required for any activity that was routine before the operation . bladder neck contracture was detected by uroflowmetry and cystoscopy in patients complaining of voiding difficulty . clinicopathologic factors were compared between the 2 groups by using student t - test for continuous variables and pearson chi - square test for categorical variables . of 112 patients , 62 patients underwent vesicourethral anastomosis with the running technique , whereas 50 patients underwent the procedure with the interrupted technique . the groups did not differ significantly in age , body mass index , prostate - specific antigen , prostate volume , or pathologic findings . the intraoperative extravasation rate was significantly lower in the running group than in the interrupted group ( 8.1% vs. 24.0% , p=0.01 ) . however , the rates of postoperative extravasation were similar for both groups ( 6.4% vs. 10.0% , p=0.12 ) . the time of anastomosis was significantly shorter in the running group than in the interrupted group ( 15.15.3 minutes vs. 19.34.6 minutes , p=0.04 ) . the duration of catheterization was also significantly shorter in the running group ( 9.03.0 days vs. 12.96.4 days , p<0.01 ) urinary retention occurred within 24 hours after urethral catheter removal in 4 patients ( 8.0% ) from the interrupted group and in 5 patients ( 8.1% ) from the running group , respectively ( p=0.36 ) . the cause of the inability to catheterize was partial distraction of the anastomosis posterior in each case . table 2 also compares the frequency of bladder neck contracture with the two techniques ; there was no significant difference between the groups ( 0% vs. 2% , p=0.76 ) . the rates of urinary continence at 3 , 6 , 9 , and 12 months after rp were similar in both groups . although most urinary extravasations are not clinically important , urinary extravasation can result in delayed catheter removal , decreased quality of life , longer hospital stays , increased costs , and increased risk of anastomotic strictures . the vesicourethral anastomosis procedure should consist of watertight closure with urethral realignment and mucosal coaptation . the interrupted vesicourethral anastomosis technique results in gaps between the suture sites than can cause extravasation and stricture formation . despite the fact that the running suture technique is theoretically more watertight , most surgeons use the interrupted technique because of the difficulty of anastomosis in open rp . previous studies have shown that using the running suture technique for open rp is technically feasible and permits safe , early catheter removal . to the best of our knowledge , this is the first study to compare perioperative outcomes of the running and interrupted techniques for rp . we found fewer instances of contrast medium extravasation during cystography in the running suture group ( 8.1% ) than in the interrupted suture group ( 10.0% ) , but without statistical significance ( p=0.12 ) . lieber et al . compared the running and interrupted suturing techniques for porcine vesicourethral anastomosis and reported greater muscle fibrosis in the interrupted suture group . compared the two techniques in 250 patients prospectively and found no significant differences in extravasations , catheterization time , or the occurrence of anastomotic strictures . however , in previous studies , postoperative cystography was not performed in all patients ; therefore , the extravasation rate may have been underestimated . even though these results were similar to our results , we suggest that the running anastomotic suture may decrease the risk of postoperative urine extravasation . one explanation for our opinion is that we found that the rate of intraoperative extravasation was lower in the running suture group ( 8.1% ) than in the interrupted suture group ( 24.0% , p=0.01 ) . a positive leak test may indicate the need for additional sutures or even complete revision of the anastomosis in the case of a major leak and can predict a leak on postoperative cystography . a previous study showed that postoperative urine extravasation was more common in patients with unsatisfactory test results than in patients with satisfactory test results . however , we performed additional sutures and kept the urethral catheter in place for longer after open rp if the result of the intraoperative leak test was not satisfactory . therefore , we suggest that the additional suture and duration of catheterization might affect the postoperative extravasation rate . in a previous study , we compared the complications of ralp with those of open rp , and our results support this view . reported extravasation to be higher in an open rp group with use of the interrupted suture technique than in the ralp group with use of the running suture technique . the time taken to complete vesicourethral anastomosis was much less in our series , although there was no significant difference in total operative time . similarly , poulakis et al . found that the operation time was 8 minutes shorter when the running suture technique was used for laparoscopic rp . furthermore , by using a nephrostomy tube , we prevented the risk of damage to the urethral stump that is usually observed when forceps are used . our running technique starts and ends at the 4 and 8 o'clock positions , respectively , thus minimizing the risk of a distraction defect at the 6 o'clock position . the intraluminal knots may promote edema in the mucosa , thereby leading to retention ; the more knots used , the greater the risk of periurethral muscle fibrosis . therefore , we anchored the anastomosis with two sutures in the 12 o'clock position and one extraluminal knot . in the study by harper and brien , the urinary retention rate was higher ( 19% ) . however , this was not due to the running technique and may have been due to postoperative edema or removing the catheter too early . another study suggested that the running suture technique did not increase the rate of urinary retention and might allow early catheter removal because earlier healing to a watertight state could lead to an earlier decrease in edema . the literature supports our findings and etiopathogenesis appears to be related to edema and postoperative bladder neck overtone . in this study , we found only one patient with bladder neck contracture in the interrupted suture group and none in the running suture group . the literature suggests that the incidence of bladder neck contracture is highly variable , ranging from 0.5% to 32% . the main risk factors for anastomotic strictures are perianastomotic urinary extravasation , blood loss , urethral ischemic damage , catheter withdrawal time , and previous transurethral resection of the prostate . the risk of ischemia is not increased by too many sutures , knots being too tight , or excessive suture tension . the rate of continence , defined as not requiring pads , was not significantly different between the two groups in our study . early recovery of continence may be delayed owing to vesicourethral urinary extravasation and there is a higher associated incidence of incontinence . however , incontinence is a complication that has been attributed to the rp itself . the present study had some limitations because the findings represent the clinical experience of a single center and a single surgeon and only a small number of patients underwent open rp . furthermore , the study design was retrospective and nonrandomized , and we suggest that well - designed prospective randomized trials that include a larger patient population are warranted . however , the baseline clinical characteristics of the patients were not significantly different between the two groups in the present study and we used the two methods under same conditions . this remains the first comparative study of the use of the two approaches in open rp . both anastomosis techniques provide similar results in terms of restoring function . however , running anastomosis may decrease the risk of intraoperative urine extravasation and had a shorter duration than the interrupted technique , without increasing the risk of urinary retention or bladder neck contracture after open rp . we recommend the running anastomosis technique for open rp because it is technically feasible and safe .
purposeto compare perioperative outcomes between running and interrupted vesicourethral anastomosis in open radical prostatectomy ( rp).materials and methodsthe medical records of 112 patients who underwent open rp for prostate cancer at our institution from 2006 to 2008 by a single surgeon were retrospectively reviewed . preoperative , intraoperative , and postoperative parameters were measured.resultsof 112 consecutive patients , 62 patients underwent vesicourethral anastomosis by use of the running technique , whereas 50 patients underwent anastomosis with the interrupted technique . the groups did not differ significantly in age , body mass index , prostate - specific antigen , prostate volume , or pathologic findings . the intraoperative extravasation rate was significantly lower in the running group ( 8.1% vs. 24.0% , p=0.01 ) . the mean anastomosis time was 15.15.3 and 19.34.6 minutes in the running and interrupted groups , respectively ( p=0.04 ) . the rates of postoperative extravasation were similar for both groups ( 6.4% vs. 10.0% , p=0.12 ) . the duration of catheterization was significantly shorter in the running group ( 9.03.0 days vs. 12.96.4 days , p<0.01 ) . the rate of urinary retention after catheter removal and the rate of bladder neck contracture were not significantly different between the two groups . the rate of urinary continence at 3 , 6 , 9 , and 12 months after rp was also similar in both groups.conclusionsboth anastomosis techniques provided similar functional results and a similar rate of postoperative urine extravasation . however , running vesicourethral anastomosis decreased the rate of intraoperative extravasation and time for anastomosis , without increasing the risk of urinary retention or bladder neck contracture .
You are an expert at summarizing long articles. Proceed to summarize the following text: the inflammation is a sequence of events that occurs in response to noxious stimuli , infection , trauma , or injury in the living tissues . the inflammation is initiated by a cascade of events including enzyme activation , mediator release , fluid extravasations , cell migration , tissue breakdown , and repair processes . these white blood cells synthesize several biomolecules and release them after injury leading to swelling and redness . prostaglandins are one of the important biomolecules , which play a key role in the induction of inflammatory response as their biosynthesis is significantly increased during inflammation . the inflammatory responses are elicited as a defense mechanism by an organism or tissues ; however , sustained inflammation can lead to undesired health effect as a consequence of interplay of various biomolecules that are secreted during the process of inflammation . the agents that contain or block inflammation may play an important role in treating pathologies associated with inflammatory reactions . natural products have a long history of use as a folk remedy for inflammatory conditions including fevers , pain , migraine , and arthritis . many of the diseases in the modern world are thought to be due to inflammation ; therefore , anti - inflammatory agents , anti - inflammatory food and food products are of great interest to contain or reduce inflammation - induced health disorders . fossil records indicate the use of natural products , especially the plants as medicine since middle paleolithic ( approximately 60,000 years ) age . the modern allopathic drugs are single active chemical molecules and target one specific pathway , whereas herbal medicines contain pleiotropic molecules that work on an orchestral approach which are able to target many elements of the complex cellular pathway . the pain and inflammatory conditions are usually managed by either steroidal ( corticosteroids ) or nonsteroidal ( aspirin ) drugs , which induce toxic side effects at different levels including allergic reactions , occasional hearing loss , and renal failure . the medicinal plants have been a major source of a wide variety of biologically active compounds for many centuries and have been used extensively in crude form or as pure isolated compounds to treat various disease conditions including inflammation . hydrocotyle umbellata and several other plants have been reported to possess anti - inflammatory activity in different study systems [ 14 , 15 ] . sonapatha or oroxylum indicum belongs to family bignoniaceae and it is characterized by brown bark and large pinnate leaves . it is a medium sized , deciduous tree , distributed in india , sri lanka , malaysia , china , thailand , philippines , and indonesia . in india , oroxylum is found in eastern and western ghats and also in the north - east regions . the existence of oroxylum indicum ( l ) vent . in natural population is highly threatened and it has been categorized as endangered medicinal plant by the government of india . the decoction of the bark is used to cure gastric ulcers and the bark paste is useful in treating mouth cancer , scabies , and other skin diseases . poultice of the bark is topically applied to treat rheumatism , sprains , inflammations , and skin diseases . apart from this , oroxylum species are reported to have a variety of medicinal properties like anticancer , antiulcer , antidysenteric , antimicrobial , and anti - inflammatory . it has been shown to be antibacterial , antioxidant , hepatoprotective , and immunomodulatory . from the above it is clear that the systematic evaluation of anti - inflammatory and analgesic activities of oroxylum indicum is lacking , which stimulated us to obtain an insight into the anti - inflammatory and analgesic activities of oroxylum indicum in swiss albino mice . the noninfected and matured stem bark of oroxylum indicum ( family : bignoniaceae ) was collected from champhai ( 23.456n latitude and 93.329e longitude ) , mizoram , india , during the month of january . the plant was identified by the department of horticulture and aromatic and medicinal plants , mizoram university , aizawl , india . the bark of o. indicum was thoroughly rinsed with clean water and shade dried at room temperature in the dark in clean and hygienic conditions . the stem bark powder of o. indicum was sequentially extracted in petroleum ether , chloroform , ethanol , and distilled water according to increase in polarity using a soxhlet apparatus until the solvents became colourless . the ethanol extract was concentrated using rotary evaporator and stored at 70c until further use . henceforth the ethanol extract of o. indicum will be referred to as oie throughout the paper . the animal care and handling were carried out according to the guidelines issued by the world health organization , geneva , switzerland , and the insa ( indian national science academy , new delhi , india ) . usually , 6-to-8-week - old healthy male swiss albino mice weighing 3035 g were selected from an inbred colony maintained under the controlled conditions of temperature ( 25 2c ) and humidity ( 5560% ) with 12 hours of light and dark cycle , respectively . the animals were housed in a sterile polypropylene cage containing paddy husk ( procured locally ) as bedding material . all animal experiments were carried out according to nih and indian national science academy , new delhi , india guidelines . the study was approved by the institutional animal ethics committee of the mizoram university , aizawl , mizoram : india vide letter number mzu / iaec/4503 . the anti - inflammatory and analgesic activities were determined by dividing the animals into the following groups . the animals of this group did not receive any treatment except the sterile physiological saline ( sps ) . the animals of this group were injected with 20 mg / kg b.wt . of diclofenac sodium the animals of this group were administered with 250 and 300 mg / kg b.wt . of ethanol the analgesic and anti - inflammatory activities were determined 30 minutes after the administration of sps or diclofenac ( dif ) or ethanol extract of oroxylum indicum . a separate experiment was conducted to determine analgesic activity of oie by the hot plate test as described earlier , where the grouping and other conditions were essentially similar to those described above . the hotplate contained metallic surface ( diameter 20 and 10 cm high ) and its temperature was set at 55c . briefly , each mouse was placed onto the hotplate and covered with a glass beaker to avoid heat loss . each mouse also acted as its own control . the time taken to lick the fore paws or jumping was recorded . the latency is defined as the reaction time taken by each mouse to respond to licking of the fore paws or jumping . the percent inhibition was calculated as follows : ( 1)posttreatment latencyspretreatment latencys100pre - treatment latencys . a separate experiment was performed to evaluate the analgesic activity by acetic acid induced writhing test , which was carried out as described earlier . the mice were administered intraperitoneally with 0.7% v / v acetic acid ( volume of acetic acid did not exceed 10 l / g b.wt . ) . immediately after acetic acid administration , the mice were individually placed into glass beakers and five min was allowed to elapse . the number of writhes produced in these animals was counted up to 30 min . for scoring purposes , a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb . a separate experiment was conducted to evaluate the analgesic activity of sonapatha ethanol extract by tail immersion test according to the procedure described elsewhere . the grouping and other conditions were essentially similar to those described above in section 2.3 . the tail immersion test was carried out in a hot water bath set at a temperature of 55 0.5c , where 3 cm of animal tail was immersed into the hot water and tail withdrawal reaction was recorded as time in seconds in all groups using a digital stopwatch . a minimum of two observations were collected for each animal in control group , immediately and 10 min after the initial reading . the tail withdrawal test was carried out in the treatment groups periodically at 0 , 0.5 , 1 , 2 , 3 , 4 , and 6 hours after administration of oie , or diclofenac . the anti - inflammatory activity was studied by xylene - induced ear edema and formalin induced paw edema in mice . a separate experiment was carried out to evaluate the anti - inflammatory activity by xylene - induced ear edema as described earlier . the grouping and other conditions were essentially similar to those described above in section 2.3 . the mice were intraperitoneally administered with either distilled water ( 10 l / g b.wt . ) or diclofenac ( 20 mg / kg b.wt . ) or oie ( 250300 mg / kg b.wt . ) . thirty minutes after administration the inner surface of right ear of each animal was applied with 0.03 ml of xylene for the induction of ear edema , whereas the left ear served as the control . fifteen minutes after the application of xylene , the mice were killed under ketamine anesthesia . circular sections of both the ears were taken , using a cork borer of 6 mm , and weighed : ( 3)inhibition ( % ) = difference in ear weight ( control)difference in ear weight ( test)difference in ear weight ( control)100 . a separate experiment was conducted to evaluate the anti - inflammatory activity by formalin induced inflammation . the grouping and other conditions were essentially similar to those described above in section 2.3 . the inflammation was produced by subaponeurotic injection of 0.1 ml of 2% formaldehyde in the right hind paw of the mice on the first and third day . the daily changes in paw size were measured by wrapping a piece of cotton thread around the paw and measuring the circumference with a meter rule . the data were analyzed by one - way anova , followed by application of tukey test ( pro 8 sro v8.0724 ( b724 ) ) , northampton , ma , usa . a p value of < 0.05 was considered to be statistically significant . the results of analgesic and anti - inflammatory activities are presented in tables 14 and figure 1 . the administration of ethanol extract ( oie ) showed a significant analgesic activity for both 250 and 300 mg / kg and 300 mg / kg exhibited the highest activity ( 62.5% inhibition ) as compared to 250 mg / kg b.wt . the positive control diclofenac showed higher analgesic activity than oie ( 76.31% inhibition ) at a dose of 20 mg / kg b.wt . ( table 1 ) . the results of acid writhing test are depicted in table 2 . administration of acetic acid to control mice produced 66.2 1.16 writhes within 30-minute observation period . pretreatment with the oie at 250 and 300 mg / kg b.wt . reduced the number of writhes up to 22.5 1.12 ( 66.0% inhibition ) and 20.8 0.74 ( 68.58% inhibition ) , respectively . the standard drug diclofenac reduced the number of writhes to 10.8 0.74 ( 83.68% inhibition ) at a dose of 20 mg / kg b.wt . the tail immersion test indicated that both the doses of the extract as well as the positive controls showed a significant inhibition in tail immersion rest when compared to the negative control ( table 3 ) . the results of the anti - inflammatory study using xylene - induced ear edema are shown in table 4 . sps treated control mice showed an increase in ear weight up to 13.98 0.60 mg and the oie administration has inhibited this weight gain by 68.097% ( 4.46 0.89 mg ) and 71.3877% ( 4.00 0.24 mg ) for 250 and 300 mg / kg b.wt . of oie , respectively ( table 4 ) . the positive control diclofenac showed 52.72% ( 6.61 0.49 mg ) inhibition at 20 mg / kg b.wt . which was lower as compared to both the doses of oie ( table 4 ) . treatment of mice with oie gradually reduced diameters of the paw with time in both the treated and positive control groups ( figure 1 ) . the oie reduced the inflammatory reactions when compared to the sps control group as indicated by the significant reduction in the paw diameter ( figure 1 ) . however , the effect was more pronounced for 300 mg / kg oie treatment ( figure 1 ) . classically inflammation is characterized by increase in the blood flow , reddening of the affected part due to increased erythrocyte accumulation and edema . physiologically inflammation results in the secretion of numerous cytokines , acute phase proteins , and recruitment of leucocytes to the site of injury . inflammation has been indicated as a major cause in the development of several diseases in humans including neurological , cardiovascular , intestinal , dental , and renal disorders . inflammation is also linked to ageing , diabetes , obesity , ankylosing spondylitis , multiple sclerosis , pancreatitis , and cancer [ 29 , 3136 ] . therefore , the present study was undertaken to evaluate the analgesic and anti - inflammatory activities of oroxylum indicum in mice . the analgesic activity of oroxylum indicum was studied by the hot plate , tail immersion , and acetic acid tests , which are standard procedures to evaluate central and peripheral nervous system acting analgesics [ 37 , 38 ] . the acetic acid is known to trigger the production of noxious substances within the peritoneum resulting in writhing response . it is a simple , rapid , and reliable model and especially suitable to evaluate peripheral type of analgesic action of any drug . the administration of oroxylum indicum extract showed a significant analgesic activity indicating that it has some analgesic effect on both the central and peripheral nervous systems as indicated by reduced pain by hot plate method and suppression of acetic acid induced writhing . several plant extracts including adhatoda vasica , acacia hydaspica , boswellia serrate , glaucium grandiflorum , and landolphia owariensis have shown analgesic activity in vivo [ 4044 ] . the tail immersion test has been used as a standard procedure to study the analgesic activity of pharmacological agents [ 45 , 46 ] , which was originally devised by . the increase in the tail withdrawal latency is a good measure of analgesia induced by any chemical agent . treatment of mice with oroxylum indicum extract increased tail withdrawal latency confirming its analgesic effects . the anti - acute inflammatory activity of any agent can be determined by xylene - induced ear edema or formalin induced paw edema tests [ 48 , 49 ] . the inflammatory phase induced pain evokes a combination of stimuli , including inflammation of peripheral tissues and mechanisms of central sensitization [ 50 , 51 ] . the central nervous system acting drugs including opioids suppress both phases equally ; however drugs that act on peripheral nervous system such as nsaids and corticosteroids only inhibit the second phase . our findings indicate that oroxylum indicum extract acts as anti - inflammatory agent as it reduced the xylene - induced ear edema as well as formalin induced paw edema in treated mice . oroxylum indicum extract has been effective in both the central and peripheral nervous systems since it was able to desensitize neurons of both central and peripheral nervous systems equally as indicated by the attenuation of pain and inflammation . many plants have been reported to possess anti - inflammatory activity in various study systems [ 15 , 52 ] . similarly , adhatoda vasica , acacia hydaspica , boswellia serrate , glaucium grandiflorum , and landolphia owariensis , harpagophytum procumbens , rosa canina , oenothera biennis , ribes nigrum , borago officinalis , zingiber officinale , nigella sativa , and folium eriobotryae have been reported to act as anti - inflammatory agents in different study systems [ 4044 , 53 , 54 ] . however , it contains flavonoids and other phenolic compounds that may have contributed to its analgesic and anti - inflammatory actions . o. indicum has been found to scavenge dpph , superoxide anion , hydroxyl , nitric oxide , and fe radicals , which are major players in eliciting the inflammatory response . apart from free radical scavenging , it may have also reduced activation of cytokines like nf-b , tnf , il-1 , and ifn. inflammation has been reported to stimulate the activation of these cytokines [ 56 , 57 ] . biochanin - a present in the root bark of o. indicum has been reported to inhibit tnf . chrysin has been isolated from the ethanol extract of o. indicum ( data not shown ) which has been reported to suppress the transcriptional activation of nf-b and cox - ii . the observed anti - inflammatory action of o. indicum may also be due to its inhibitory action on cyclooxygenase which is involved in prostaglandin synthesis . our study demonstrates that the o. indicum acts as an analgesic and anti - inflammatory agent . the analgesic and anti - inflammatory activities of o. indicum may be due to its ability to neutralize free radicals which are the main players in inflammation . it may have also suppressed the activation of proinflammatory cytokines including nf-b , tnf , il-1 , and ifn and the activity of cyclooxygenase enzymes which are involved in inflammation . the anti - inflammatory and analgesic activities of o. indicum may be due to the presence of flavonoids and other polyphenols . the o. indicum may be used to reduce inflammation ; however , further studies are required to understand molecular mechanisms of action against inflammation .
inflammation is all a pervasive phenomenon , which is elicited by the body in response to obnoxious stimuli as a protective measure . however , sustained inflammation leads to several diseases including cancer . therefore it is necessary to neutralize inflammation . sonapatha ( oroxylum indicum ) , a medicinal plant , is traditionally used as a medicine in ayurveda and other folk systems of medicine . it is commonly used to treat inflammatory diseases including rheumatoid arthritis and asthma . despite this fact its anti - inflammatory and analgesic effects are not evaluated scientifically . therefore , the anti - inflammatory and analgesic activities of sonapatha ( oroxylum indicum ) were studied in swiss albino mice by different methods . the hot plate , acetic acid , and tail immersion tests were used to evaluate the analgesic activity whereas xylene - induced ear edema and formalin induced paw edema tests were used to study the anti - inflammatory activity of sonapatha . the administration of mice with 250 and 300 mg / kg b.wt . of o. indicum reduced pain and inflammation indicating that sonapatha possesses analgesic and anti - inflammatory activities . the maximum analgesic and anti - inflammatory activities were observed in mice receiving 300 mg / kg b.wt . of o. indicum ethanol extract . our study indicates that o. indicum possesses both anti - inflammatory and analgesic activities and it may be useful as an anti - inflammatory agent in the inflammation related disorders .
You are an expert at summarizing long articles. Proceed to summarize the following text: what has brain imaging found ? just over 10 years ago , a landmark study used anatomic mri to compare 152 children with adhd to a group of 139 children with no symptoms . the adhd group showed a slight reduction about 3 percent in total brain volume.5 this did not mean the children with adhd were less intelligent ; they were as intelligent as the comparison children in the study . rather , it suggested that severe , impairing problems with attention , impulse , and activity control are associated with differences in brain structure . since this study , marked advances in acquiring and analyzing brain images have enabled researchers to pinpoint the brain regions most tightly linked with adhd . these advances allow the identification of structural differences in the hubs of networks that most strongly contribute to challenges with control of attention and movement . many studies have defined brain - volume change in terms of voxels . the key components or hubs of the networks that support the control of action and attention are shown . these include ( a ) regions of the prefrontal cortex ( particularly the dorsolateral cortex , and the midline cingulate cortex , which is not shown ) ; ( b ) parts of the striatum ; ( c ) the cerebellum , particularly the midline vermis . when we pool the results of these studies , the aggregate findings point to key hubs that might be important in the control of attention . the first candidate hub lies in the striatum , a deep brain structure made up of two substructures , the caudate and the putamen . the striatum , which lies near the center of each hemisphere , interacts richly with multiple other brain regions . it is a hub in networks supporting many cognitive skills , such as the flexible control of attention and motor planning . the effect is more prominent on the right side of the putamen and the anterior parts of the caudate . many individuals with adhd find attention - control and motor - planning skills particularly challenging . more recent work suggests that changes to the striatum apply not only to its volume , but also to its surface contours , with reports of indentation in parts of the striatum in groups with adhd.26 at an even deeper level in the striatum , images show changes in the density of receptors for dopamine , which is one of the chemicals in the brain that is important in communication between cells.29 a second set of candidate hubs has been localized to the prefrontal cortex . this set emerged partly from studies that mapped both cortical thickness and surface area at thousands of points across the brain . most of these studies indicate that the dorsolateral prefrontal cortex is slightly thinner in groups with adhd compared to those without the condition.1 , 2 , 17 , 23 this frontal region is a central hub in the networks controlling the most complex executive functions , such as allocation of attention , planning , and decisionmaking . a second structurally altered hub lies in the cingulate cortex , which is pivotal in monitoring the environment and adjusting behavior in response to feedback . tellingly , both of these frontal areas belong to networks that include parts of the striatum . a child s environment has an exquisite temporal structure ; successful navigation requires the ability to process information that changes extremely quickly on the order of milliseconds . 6 , 18 , 21 , 22 , 25 , 27 for example , a conversation requires the ability to judge exactly when someone is about to stop speaking and then to respond within an appropriate time period . a child may appear impulsive because they fail to judge exactly the correct amount of time needed before responding to a teacher , for example , and interrupt . the cerebellum is a critical part of the networks responsible for such temporal information processing . decreased volume of the cerebellum has been found in groups with adhd , compared to groups without adhd.3 , 16 the midline section of the cerebellum called the vermis has been most strongly linked with attention problems . notably , the vermis is richly interconnected with the cortical hubs mentioned earlier . in short three hubs emerge as important : centers in the prefrontal cortex , the striatum , and the cerebellum . for example , one hub might be able to compensate for another , a point we will return to later . it leverages the fact that different tissues and regions can produce a different signal when placed in a strong magnetic field . scientists once established these boundaries by tracing around entire structures with their hands , but over time they have increasingly used objective computational approaches to define structures at a much finer level of resolution . for example , in one approach , scientists divide the brain into hundreds of thousands of tiny boxes , or voxels ( mentioned earlier ) , and measure the volume of each voxel . in another , they look at the thickness of the cortex at hundreds of thousands of points across the brain . both approaches afford exquisite precision in mapping anatomical differences . white matter tracts , which often connect different brain regions , have physical properties that can be measured by mri . water molecules in the brain move randomly unless they are constrained by a physical barrier . white matter tracts , or , more precisely , the myelin sheaths that form tracts , are one such barrier . these tracts can be mapped using a form of mri called diffusion tensor imaging ( dti ) , which enables us to better understand the connections between brain regions . when describing the findings , it is important to stress that the brain - structure differences emerge from studies that compare one group with adhd against a group without it . on average , the size of certain brain structures however , the size of brain structures varies greatly within both groups , and the size of brain structures also overlaps significantly between the groups . for example , the volume of a structure called the striatum is lower , on average , in groups of people who have adhd than it is in groups of people who do not have adhd . this means that the distribution ( or the spread ) of striatal volumes in adhd is shifted , compared to the distribution of areas in the comparison or control group . there is , however , still much overlap between the distributions . in other words , the volume of an individual s striatum is insufficient information for a diagnosis ; the diagnosis is still based on a careful assessment that includes feedback from family members and teachers . nonetheless , the robust group differences found in neuroimaging studies give us invaluable insights into the neurobiology of inattention , impulsivity , and hyperactivity . researchers are benefiting from the emerging picture of group - level structural change in brain regions that are pivotal in the cognitive processes most affected in adhd . carrying information quickly and efficiently between hubs in a network is a prerequisite for optimal brain functioning . using techniques such as dti scientists debate about the exact physical properties that dti captures , but broadly speaking , one measure some themes emerge when we pool the results of dti studies comparing groups of individuals with and without adhd . 13 , 28 first , fractional anisotropy is lower in some regions of the brain in groups with adhd compared to groups without the condition . these changes are prominent , but they are not confined to the tracts that link the hubs mentioned earlier . to give a specific example , reduction in fractional anisotropy localizes to a region near the tracts that connect the cerebellum with prefrontal cortical hubs important for motor control . another region that shows change contains the white matter tracts that connect different cortical hubs ( the superior longitudinal fasciculus ) . accelerating the development of new methods to map brain structural connectivity is the human connectome project , a five - year project sponsored by 16 components of the national institutes of health and split between two consortia of research institutions . the project is the first large - scale attempt to collect and share data of a scope and level of detail sufficient to begin the process of addressing fundamental questions about human connectional anatomy and variation , with the hope of gaining a richer understanding of the connectivity problems that underpin challenges in attention and impulse control . are these brain structural changes the result , rather than the cause , of problems with inattention , impulsivity , and hyperactivity ? evidence from studies on families indicates that brain anatomic changes are not merely the result of having symptoms of adhd , but rather they are a causative factor . in these studies , siblings with and without adhd the studies indicate that changes in brain structures in unaffected siblings that resemble the structural changes in siblings with adhd can not be due to adhd symptoms , as the unaffected siblings have no symptoms . rather , the brain changes reflect familial , likely genetic , influences on brain anatomy . one study demonstrated that children with adhd and their unaffected siblings share similar structural brain differences , including a slight reduction in the volume of the prefrontal cortex.9 another study found white matter tract structure changes in both individuals with adhd and siblings without adhd.15 similar family studies have long been used to isolate genes that contribute to mental - health problems . an exciting future direction is to adopt this approach in neuroimaging by identifying the brain changes that track attention problems within families . such work is particularly compelling given the possibility of sequencing the genome that is , spelling out the entire genetic code of an individual . harnessing the power of both genomic sequencing and rapidly advancing neuroimaging techniques will yield great insights into the mechanisms underpinning the control of attention and action and why they sometimes go awry . think back to susan , the child whose problem with staying focused was impacting her ability to learn . how do children with problems similar to susan s fare as they grow up ? several large studies follow children with a diagnosis of adhd into adulthood.24 full - blown adhd persists around 20 percent of the time . while a further 50 percent have symptoms that impair day - to - day living , they do not have enough symptoms to warrant a formal diagnosis . generally , hyperactivity and impulsivity tend to improve more than inattention does . as with many challenges of childhood understanding the brain changes or trajectories that underpin these variables could help us devise treatments to keep brain development on track . children with adhd underwent repeated assessment of their symptoms along with mris as they grew into adulthood.24 this approach allowed researchers to link the trajectories of brain development with adult outcomes . some differences in trajectories emerged in a prefrontal cortical region mentioned earlier : the cingulate cortex . this region is a key hub in a network that keeps an individual on task and monitors the environment for relevant information.19 in the study , individuals whose attention improved started with a thinner cortex in childhood , but the cortex eventually grew to the same thickness as a comparison group that never had adhd . by contrast , those who had persistent inattention into adulthood did not show this convergence ; rather , their cortices showed a fixed difference . there was a great deal of variability in these trajectories , but overall differences in the development of an attention hub were associated with later outcome . movies provide more information than snapshots do ; likewise , developmental differences important for the control of action and attention can often be captured better by longitudinal than cross - sectional data.14 thinking of the brain as a developing network might inspire new approaches to help people overcome impairing problems with attention and impulsivity . perhaps intact hubs in a network can be recruited to compensate for suboptimal performance of other networks.7 or perhaps through techniques such as cognitive - skill training , people with adhd can improve communication between key hubs.12 our emphasis on the importance of taking a developmental perspective underscores the brain s capacity for beneficial change . it is exciting to think how the enormous potential for brain plasticity in childhood and adulthood could be harnessed to improve attention skills and to boost motor and cognitive control .
editor s note : estimates of children struggling with attention - deficit / hyperactivity disorder ( adhd ) vary , but the centers for disease control puts the number at a stunningly high 25 percent . whatever the number , adhd affects too many children at school , at home , and with their peers , and often persists into adulthood . the cause is as yet unknown , although genetic factors and their interaction with the environment are known to be pivotal . ten years ago a landmark study showed that the structure of the brains of children with adhd differs from that of unaffected children . since that study , enhancements in imaging have given researchers a better look at key hubs in the brain and how they network advances that could prove useful in the control and treatment of adhd in both children and adults .
You are an expert at summarizing long articles. Proceed to summarize the following text: when a stone obstructs the gallbladder ( gb ) neck or cystic duct , an acute inflammation process can occur . the prevalence of cholelithiasis , presence of stones in the gb , is about 1015% in the general population . among those with cholilithiasis , overall , acute cholecystitis will develop in up to 30% of patients with cholilithiasis . however , acute cholecystitis develops in men more frequently than would be expected from the relative prevalence of gallstones ( about half that in women ) , and cholecystitis tends to be more severe in men . in patients with diabetes , acute cholecystitis seems to develop more frequently than in patients without diabetes , and such patients are more likely to have complications of acute cholecystitis when it occurs . the symptoms include right upper quadrant abdominal pain , fever , nausea and vomiting , loss of appetite , and mild jaundice . the clinical symptoms and laboratory findings for acute cholecystitis are nonspecific and they can also be found in other conditions . in elderly patients , delays in diagnosis are common because physical examination and laboratory indexes may be normal . a 69-year old male , who underwent ascending aortic aneurysm repair with no past medical history of alcohol or tobacco abuse , hypertension , diabetes mellitus , obesity and peptic ulcer disease on day twelve postoperatively developed low - grade fever and leukocytosis . his white blood cell count was 16.60 ( normal 4.810.8 billion cells / l ) . a chest computed tomography ( ct ) revealed a periaortic hematoma which represents a postsurgical change from aortic aneurysm repair , and a small pericardial effusion . an ultrasound ( us ) examination of the abdomen was done and it showed cholelithiasis and gb sludge without evidence of cholecystitis . after this , a positron emission tomography ( pet)/ct was ordered to look for any metabolic activity in the periaortic hematoma to rule out infection . the fluorodeoxyglucose ( fdg)-pet / ct examination found no abnormal fdg activity in the periaortic hematoma to indicate infection . there was an incidental finding of intensely increased fdg activity within the distended gb wall , with associated mild pericholecystic fat stranding and stones in the gb neck [ figure 1 ] . he underwent cholecystectomy and pathology confirmed the diagnosis as acute cholecystitis . a 69-year - old male developed low - grade fevers and leukocytosis , after ascending aortic aneurysm repair with aortic valve replacement . ( a ) fluorodeoxyglucose - positron emission tomography / computed tomography maximum intensity projection image shows no signs of infection around the aorta but increased metabolic activity around the gallbladder ( arrow ) . ( b ) axial view computed tomography image shows a distended gallbladder with mild pericholecystic fat stranding ( arrow ) . ( c ) fluorodeoxyglucose positron emission tomography image and ( d ) hybrid fluorodeoxyglucose - positron emission tomography / computed tomography , axial views , show increased tracer uptake in the wall of the gallbladder , with no uptake in the lumen ( arrows ) . to the best of our knowledge , there are only two prior reports in the english language literature that mention the use of fdg - pet / ct to image acute calculous cholecystitis . our paper is unique ; in that , we did not intend to diagnose acute cholecystitis with fdg - pet / ct . the abdominal us is often the first step in the workup for suspected acute cholecystitis because it is safe , widely available , and inexpensive . stogryn et al . , reported that us has a low negative predictive value and it can underdiagnose acute cholecystitis . in the current case , the us examination found cholilithiasis but no signs of cholecystitis . a prospective study by bingener et al . , found that the sensitivity and specificity of us were 54% and 67% , respectively . the same study reported that the us 's ability to predict acute cholecystitis in patients with clinical symptoms appears limited . age , diabetes mellitus , and pain medications can potentially mask a sonographic murphy sign . our patient was a 69-year - old diabetic who was on pain medications after the surgery . with us , a gallstone may or may not be visualized within the gb neck or cystic duct . the patient must be in the prone or left lateral decubitus position , to better visualize a stone impacted in the gb neck or cystic duct . radionuclide scanning using technetium - labeled hepato iminodiacetic acid ( hida ) is usually ordered after a normal us to exclude acute cholecystitis . riyad et al . , reported that hida scan is very useful in early diagnosis of biliary pain in patients who have a normal abdominal us . however , our patient had no complaints of abdominal pain , and acute cholecystitis was not suspected at first and hida scan was not ordered . a significant fraction of mixed , cholesterol or pigment stones are similar in attenuation to bile and may not be detected on ct . in the present case , our patient had an almost normal ct except for postsurgical changes and a small pericardial effusion . thus , acute cholecystitis can not be ruled out in the absence of ct findings . in a mild form or early manifestation of acute cholecystitis , the ct may be normal and inflammatory changes may be absent on ct in early stages of acute cholecystitis . pet / ct provides both anatomical and functional imaging information and it can scan the whole body in one examination everywhere to assess for possible complications after the surgery . since it relies on detecting the changes in metabolic activity of tissues , it can detect pathology even before anatomic changes are apparent . in acute cholecystitis , there is an increased blood flow from the cystic artery to the gb wall . fdg is taken up by activated inflammatory cells in the gb wall . in the current case , there was an increased fdg uptake in the gb wall , with no uptake in the lumen . this is called the rim sign , and the description is consistent with the previously reported findings of acute cholecystitis on fdg - pet / ct . in contrast , in gb cancers , the fdg uptake is either focal or it fills up the gb , with no photopenic areas . thus , fdg - pet / ct can distinguish between benign and malignant lesions in the gb . finally , the patients can undergo multiple imaging studies for evaluation of acute cholecystitis before cholecystectomy . our case has shown that fdg - pet / ct can eliminate the need for multiple examinations and the delay in treatment . the present case report has shown that both us and ct can miss acute cholecystitis . fdg - pet / ct can diagnose acute cholecystitis in patients with nonspecific clinical symptoms and non - specific imaging results . fdg - pet / ct has a promising role in the management of acute cholecystitis .
we present a case of a 69-year - old patient who underwent ascending aortic aneurysm repair with aortic valve replacement . on postsurgical day 12 , he developed leukocytosis and low - grade fevers . the chest computed tomography ( ct ) showed a periaortic hematoma which represents a postsurgical change from aortic aneurysm repair , and a small pericardial effusion . the abdominal ultrasound showed cholelithiasis without any sign of cholecystitis . finally , a fluorodeoxyglucose ( fdg)-positron emission tomography ( pet)/ct examination was ordered to find the cause of fever of unknown origin , and it showed increased fdg uptake in the gallbladder wall , with no uptake in the lumen . fdg - pet / ct can diagnose acute cholecystitis in patients with nonspecific clinical symptoms and laboratory results .
You are an expert at summarizing long articles. Proceed to summarize the following text: lymphangiomas are rare benign tumors that are preferentially located in the head and neck in children ( 1 ) . lymphangiomas in the peritoneal cavity are extremely rare occurring most commonly in the mesentery , followed by the omentum , mesocolon , and retroperitoneum . the etiology is unclear , but they are considered primarily to congenital in origin ( 2 ) . we report the case of a young woman with appendicular pain caused by a cystic lymphangioma of the right iliac fossa : a rare but possible differential diagnosis . a 17-year - old female patient presented with a dull abdominal pain in the right iliac fossa and flank of a 1-month duration . an abdominal examination revealed hyperactive bowel sounds and tenderness during palpation in the central abdominal quadrant and right iliac fossa in mc burney point . neoplastic markers ( afp , cea , ca 19 - 9 , ca 15 - 3 , ca125 , tps ) were negative . axial and coronal t2-weighted tse images showed the presence of a fluid - filled cystic lesion in the retrocaecl adipose tissue . an ultrasonographic and ct scan of the abdomen revealed the presence of a cystic mass ( max diameter 60 mm ) in the retrocaecal adipose tissue that was in proximity of the caecum , appendix and ileum . in addition , the ct scan revealed the presence of multiple hypodense formations in the body and tail of pancreas . during the hospital the exam revealed in the retrocaecl adipose tissue the presence of a fluid - filled formation with a diameter of 60 mm that appeared indissociable from the caecum for the presence of a tissue connection ( fig 1 ) . other cystic lesions ( communicating with pancreatic duct ) were present in the whole pancreas and in the right kidney . fluid - filled cystic lymphangioma with polycyclic edge and internal septa ( intraoperative specimen ) the patient underwent exploratory surgery : at laparotomy a yellowish oval formation with a diameter of 6 cm was found arising from the mesentery of terminal ileum with partial adhesion of the appendix . the lesion had a cystic aspect and was fluid - filled ( fig 2 ) . the patient has been followed - up for two years with semestral clinical examination and abdominal eco scan and no recurrence occurred . intra - abdominal mesenteric lymphangioma is an uncommon tumor that usually manifestes in early adulthood . this tumor has an incidence of approximately 1/20,000 and 1/250,000 hospital admissions , and it is more frequent in men than women with a m / f ratio of 3:1 ( 3 ) . in intraperitoneal sites , the small bowel mesentery ( 70% ) is the commonest localization , with 5060% of all cysts located in the ileal mesentery . ( 4 ) the etiology of mesenteric lymphangioma is considered to be congenital , with abnormal embryonic development of the lymphatic system causing sequestration of lymphatic tissue ( 1 ) : infact in our patient also other cystic lesions ( kidney and pancreas ) were found at abdomen ct / mri . radiological investigations are a useful diagnostic tool , but definitive diagnosis is confirmed by histopathology after a complete surgical resection . ( 5 ) at immunochemistry : the lining mesothelial cells are immunoreactive for cytokeratin and negative for factor viiiis . double staining with prox1 and cd31 is the most reliable method for characterizing lymphangioma endothelial cells.(6 )
pain of the right iliac fossa in young girls can relate to several pathologies of the gastrointestinal and genitor - urinary tract . on the other hand , lymphangiomas in the peritoneal cavity are extremely rare . we report the case of a young woman with pain in the right iliac fossa caused by a cystic lymphangioma . a 17-year - old caucasian female patient presented with abdominal pain in the right iliac fossa and flank of a 1-month duration . the patient was apyrexic and laboratory tests were negative . ct / mri of abdomen showed a cystic mass ( max diameter 60 mm ) in the retrocaecl adipose tissue in connection with the caecum and appendix . the patient underwent a total excision of the lesion and appendicectomy . histopathological examination was consistent with the diagnosis of mesenteric lymphangioma . this case shows a rare but possible cause of right iliac fossa pain in young women to be considered in the differential diagnosis .
You are an expert at summarizing long articles. Proceed to summarize the following text: a 56-year - old female presented with a palpable skin nodule on the anterior chest wall . the nodule was well - defined , ovoid , pale tan , and rubbery firm , and measured 2.21.51.5 cm . on microscopic examination , the nodule was well - circumscribed and composed of slender spindle cells arranged in loose storiform or short fascicular patterns . the tumor cells had wavy nuclei and long streamer - like cytoplasmic processes ( fig . an electron microscopic study failed to reveal any detailed findings due to inappropriately preserved formalin - fixed paraffin - embedded tissue . a 53-year - old male visited our out - patient clinic for the evaluation of the posterior mediastinal mass , incidentally found during a regular health check - up . thoracoscopic excision of the mass was performed . the gross specimen consisted of multiple fragments of pale tan , translucent , and relatively firm the majority of the mass was composed of spindle cells arranged in short fascicular or parallel cords in a fibromyxoid matrix as seen in soft tissue perineuriomas . another spindle cell area was noted , manifesting as a typical schwannoma , including antoni a and b areas , nuclear palisading , and hyalinized blood vessels ( fig . the perineurioma - like area of the mediastinal tumor was diffusely and strongly positive for ema and claudin-1 , but negative for s-100 protein ( fig . however , the tumor cells were evenly and strongly immunoreactive for s-100 protein in the schwannoma - looking area , but did not stain for ema or claudin-1 ( fig . specimens were sampled separately from the two representative areas of the formalin - fixed paraffin - embedded block for ultrastructural studies . the cells of the perineurioma - like area showed slender and long - tapered processes containing pinocytic vesicles and discontinuous external lamina ( fig . in contrast , the schwannoma - looking area exhibited tumor cells with attenuated cell processes invested by continuous basal lamina ( fig . a 56-year - old female presented with a palpable skin nodule on the anterior chest wall . the nodule was well - defined , ovoid , pale tan , and rubbery firm , and measured 2.21.51.5 cm . on microscopic examination , the nodule was well - circumscribed and composed of slender spindle cells arranged in loose storiform or short fascicular patterns . the tumor cells had wavy nuclei and long streamer - like cytoplasmic processes ( fig . an electron microscopic study failed to reveal any detailed findings due to inappropriately preserved formalin - fixed paraffin - embedded tissue . a 53-year - old male visited our out - patient clinic for the evaluation of the posterior mediastinal mass , incidentally found during a regular health check - up . thoracoscopic excision of the mass was performed . the gross specimen consisted of multiple fragments of pale tan , translucent , and relatively firm the majority of the mass was composed of spindle cells arranged in short fascicular or parallel cords in a fibromyxoid matrix as seen in soft tissue perineuriomas . another spindle cell area was noted , manifesting as a typical schwannoma , including antoni a and b areas , nuclear palisading , and hyalinized blood vessels ( fig . the perineurioma - like area of the mediastinal tumor was diffusely and strongly positive for ema and claudin-1 , but negative for s-100 protein ( fig . 2b , c ) . however , the tumor cells were evenly and strongly immunoreactive for s-100 protein in the schwannoma - looking area , but did not stain for ema or claudin-1 ( fig . specimens were sampled separately from the two representative areas of the formalin - fixed paraffin - embedded block for ultrastructural studies . the cells of the perineurioma - like area showed slender and long - tapered processes containing pinocytic vesicles and discontinuous external lamina ( fig . in contrast , the schwannoma - looking area exhibited tumor cells with attenuated cell processes invested by continuous basal lamina ( fig . schwannomas are composed of schwann cells and have characteristic cellular features , including antoni a and b areas , nuclear palisadings ( verocay bodies ) , and hyalinized vessels with perivascular hemosiderin depositions . neurofibromas consist of an admixture of schwann cells , fibroblasts , perineurial cells , and scattered axons.1 perineuriomas are exclusively composed of differentiated perineurial cells . perineuriomas can be divided into four categories , rare intraneural perineuriomas , more common soft tissue ( extraneural ) perineuriomas , and sclerosing and reticular variants.2,6 intraneural perineuriomas , formerly known as ' localized hypertrophic neuropathy ' usually occur in the extremities of young individuals.1 they microscopically show concentric proliferation of perineurial cells around axons and schwann cells , forming " onion bulbs " that stain positive for ema.1 sclerosing perineuriomas occur exclusively in the acral areas of young adults and are characterized by cords of bland epithelioid cells embedded in sclerotic collagen.1 reticular perineuriomas are a recently recognized variant of perineuriomas , which are characterized by a predominantly lace - like or reticular growth pattern composed of fusiform cells with bipolar cytoplasmic processes.5 soft tissue perineuriomas primarily affect adults and involve the superficial soft tissues of the extremities and trunk ; however , approximately 30% can develop in deep soft tissue and , rarely , visceral locations.2 soft tissue perineuriomas show microscopic proliferation of spindle cells with wavy nuclei and elongated bipolar cytoplasmic processes arranged in a storiform or short fascicular pattern.1 perineurioimas are usually positive for ema and can also express claudin-1 , glut-1 , and cd34.7 electron microscopy findings are most useful for diagnosing perineuromas , which are characterized by showing perineurial cells with slender bipolar cytoplasmic processes containing pinocytic vesicles and discontinuous external lamina.1 they generally behave in a benign fashion , and surgical removal is curative.1 although the tumors are not associated with neurofibromatosis type 1 or 2 , a case of soft tissue perineurioma in a patient with neurofibromatosis type 2 has been reported.3 additionally , some studies have described that fluorescence in situ hybridization and molecular analysis demonstrate deletions or point mutations on the chromosome 22q11 in the region of the nf2 gene and chromosome 10 . these findings suggested that there might be diverse genetic mechanisms underlying the development of perineuriomas.6 these benign pnsts are currently distinct entities . these include hybrid schwannoma - neurofibromas , hybrid schwannoma - perineuriomas , and hybrid neurofibroma - perineuriomas.4,5,7 feany et al.4 and hornick et al.5 reported nine cases of schwannomas and neurofibromas hybrids and 42 cases of hybrid schwannomas and perineuriomas . they reported that hybrid tumors have well - documented histological differences with immunohistochemical and electron microscopic features and suggested that the pathogenesis of the diverse differentiation remains unknown , but could result from incomplete differentiation or genetic alteration of primitive tumor cells , or in response to a localized microenvironmental change.4,5,7 hybrid tumors usually arise in the dermis and subcutis over a wide range of age and anatomical distribution and seem to pursue a benign course.5 nuclear atypia is relatively common in the hybrid tumors , which seems degenerative in nature , including smudged chromatin and occasional nuclear pseudoinclusions . the differential diagnoses of the hybrid tumors may include other benign nerve sheath tumors and low grade malignant peripheral nerve sheath tumors ( mpnst ) . mpnst should be considered , if nuclear atypia is prominent.5 as information and clinical follow - up data on hybrid tumors are limited , additional studies are needed to understand the pathogenesis and clinicopathological significance of hybrid tumors . in conclusion , we described two interesting cases of a pure form of soft tissue perineurioma , which stained positively for ema and claudin-1 , and a hybrid perineurioma and schwannoma documented by immunohistochemical and ultrastructural features . to our knowledge , this is the first report of a hybrid peripheral nerve sheath tumor in korea .
perineuriomas are composed of differentiated perineurial cells . perineuriomas have been recently recognized by the immunoreactivity for epithelial membrane antigen ( ema ) . microscopically , perineuriomas show proliferation of spindle cells with wavy nuclei and delicate elongated bipolar cytoplasmic processes . the tumor cells are usually negative for the s-100 protein . ultrastructurally , perineurial cells reveal slender , nontapered processes containing pinocytic vesicles and discontinuous basal lamina . interestingly , hybrid tumors of benign peripheral nerve sheath tumor ( pnst ) have been recently reported by using immunohistochemical and ultrastructural investigations . herein , we report a case of soft tissue perineurioma arising in the skin of a 56-year - old female ; another case of a hybrid tumor of perineurioma and schwannoma in the posterior mediastinum occurred in a 53-year - old male , which is the first case of the hybrid pnst tumor reported in korea .
You are an expert at summarizing long articles. Proceed to summarize the following text: physicians have shown great concern on the relationship between lymphoma and inflammatory bowel disease ( ibd ) over the years . in 1928 , bargen described initial report of lymphosarcoma in a patient with ulcerative colitis . since then , numerous case reports and meta - analyses have been presented , but composite lymphoma in ibd patients had not previously been reported . composite lymphoma is a rare malignancy and defined as the presence of two or more distinct cytological and histological variant of lymphoma in the same tissue . this combination consists of hodgkin lymphoma ( hl ) with non - hodgkin lymphoma ( nhl ) or b - cell nhl with t -cell nhl , or two distinct subtype of b - cell or t - cell nhl . in this study , we present a case of a composite diffuse large b - cell lymphoma and nodular sclerosing hl in para - aortic lymph node and spleen in a 45-year- old female patient diagnosed with ulcerative colitis . the patient was a 45-year - old female with a 2-year history of left - sided ulcerative colitis . only mesalamine was responsible for keeping the disease under control and no other immunosuppressive agents such as azathioprine and anti tnf were administered . the patient was referred to hematologic ward with a one - month history of malaise , weakness and fatigue . upon physical examination , hematologic findings consisted of pancytopenia , elevated erythrocyte sedimentation rate ( esr ) and lactate dehydrogenase ( ldh ) . results of renal and liver function tests , electrolytes , urine analysis and hiv test were within the normal range . contrast - enhanced computed tomography scan of the chest and abdominopelvic regions showed lymphadenopathy of middle and posterior mediastinum , para - aortic lymphadenopathy and splenomegaly with heterogeneous density ( fig.1 ) . to achieve a definite diagnosis , splenectomy , excisional biopsy of para - aortic lymph node , aspiration and biopsy of marrow histomorphological evaluation of the spleen and para aortic lymph node ( fig : 2 ) show focal involvement by classical hodgkin s lymphoma and nodular sclerosis variant , wherein typical nodules are seen in which the lacunar cells and classic red - sternberg ( r - s ) cells are surrounded by small mature lymphocytes with a few eosinophils and plasma cells . however , in other areas , diffuse sheets of centroblastic and ( less ) immunoblastic cells are also evident , which is in keeping with diffuse large b - cell lymphoma ( dlbcl ) . 3 ) , the lacunar cells and r - s cells in the hl component show negative leukocyte common antigen ( lca ) , cd20 and positive ki - a10 ( one of the hl associated markers ) although cd30 and cd15 are negative . accordingly , there is strong positivity of lca and cd20 in the dlbcl component . treatment of composite lymphoma depends on histological grading of components and is towards the high grade histology . the patient was treated with r - chop- based chemotherapy ( rituximab , cyclophosphamide , adriamycin , vincristine and prednisolone ) . after eight courses of chemotherapy with r - chop and one course of high - dose methotrexate , complete remission was achieved . the patient was a 45-year - old female with a 2-year history of left - sided ulcerative colitis . only mesalamine was responsible for keeping the disease under control and no other immunosuppressive agents such as azathioprine and anti tnf were administered . the patient was referred to hematologic ward with a one - month history of malaise , weakness and fatigue . upon physical examination , hematologic findings consisted of pancytopenia , elevated erythrocyte sedimentation rate ( esr ) and lactate dehydrogenase ( ldh ) . results of renal and liver function tests , electrolytes , urine analysis and hiv test were within the normal range . contrast - enhanced computed tomography scan of the chest and abdominopelvic regions showed lymphadenopathy of middle and posterior mediastinum , para - aortic lymphadenopathy and splenomegaly with heterogeneous density ( fig.1 ) . to achieve a definite diagnosis , splenectomy , excisional biopsy of para - aortic lymph node , aspiration and biopsy of marrow histomorphological evaluation of the spleen and para aortic lymph node ( fig : 2 ) show focal involvement by classical hodgkin s lymphoma and nodular sclerosis variant , wherein typical nodules are seen in which the lacunar cells and classic red - sternberg ( r - s ) cells are surrounded by small mature lymphocytes with a few eosinophils and plasma cells . however , in other areas , diffuse sheets of centroblastic and ( less ) immunoblastic cells are also evident , which is in keeping with diffuse large b - cell lymphoma ( dlbcl ) . 3 ) , the lacunar cells and r - s cells in the hl component show negative leukocyte common antigen ( lca ) , cd20 and positive ki - a10 ( one of the hl associated markers ) although cd30 and cd15 are negative . accordingly , there is strong positivity of lca and cd20 in the dlbcl component . treatment of composite lymphoma depends on histological grading of components and is towards the high grade histology . the patient was treated with r - chop- based chemotherapy ( rituximab , cyclophosphamide , adriamycin , vincristine and prednisolone ) . after eight courses of chemotherapy with r - chop and one course of high - dose methotrexate , complete remission was achieved . inflammatory bowel disease is a chronic disorder of gastrointestinal tract including two main diseases : ulcerative colitis and crohn s disease . the etiology of these diseases is unknown , but evidence suggests that genetically susceptible patients immune dysfunction triggered by environmental factors is responsible for disease . the risk of lymphoma in ibd patients has been a great concern in the medicine for many years . environmental , genetic , infectious and iatrogenic factors in the patients with inflammatory bowel disease can predispose them to increase the risk of lymphoma . overall risk of lymphoma in ibd patients is not higher than general population , but the basic question is whether treatment with immunomedulatory agents can lead to an increased risk of lymphoma . the use of azathioprine /6-mercaptopurine increases the incidence of lymphoma in patients with inflammatory bowel disease although usefulness of azathioprine therapy outweigh the risk of lymphoma has been suggested in decision analysis model . ebv in the patients with ibd who received azathioprine/6-mercaptopurine associated with increased risk of lymphoma . methotrexate is another immunomedulatory drug which is used as alternative treatment in moderate to severe crohn s disease . unlike azathioprine , there is no repeated and reliable data for the risk of lymphoma in ibd patients treated with methotrexate . in the cesame study conducted by beaugerie et al . , ( 2009 ) some studies like those conducted on rheumatoid arthritis provide some information on the effect of methotrexate . in these studies , there was no increase in the risk of lymphoma among patients treated with methotrexate . at that time , anti - tnf agents such as infliximab were approved for the treatment of crohn s disease . there is a great concern over safety of drugs ; moreover , the assessment of specific risk of these agents on lymphoma development is difficult because most patients had a previous exposure to methotrexate and azathioprine . a comprehensive meta - analysis that was conducted by siegel et al . , ( 2009 ) showed that the rate of nhl in patients with crohn s disease who treated with anti - tnf agents in combination with immunomedulators was not increased . non - hodgkin s lymphoma , particularly diffuse large b - cell lymphoma , appears to be more common than other types of lymphoma in the patients with inflammatory bowel disease . she had no exposure to azathioprine , methotrexate or anti tnf agents . despite the large number of ibd patients with lymphoma , the incidence rate of composite lymphoma , known as rare malignancy , is estimated to be 1 to 4.7% of all other types of lymphoma . in contrast , the combination of non - hodgkin s lymphoma and hodgkin s disease rarely occurs . nodular lymphocyte - predominant hodgkin s lymphoma and diffuse b - cell lymphoma are more frequently found among these combinations .
a 45-year - old female patient with a diagnosis of ulcerative colitis complicated with composite lymphoma in the spleen and para - aortic lymph node presented with a one - month history of malaise , weakness and fatigue . only mesalamine kept ulcerative colitis under control . in physical examination , splenomegaly was revealed and pancytopenia was obtained from laboratory data . computed tomography scan revealed para - aortic mediastinal lymphadenopathy with splenomegaly . splenectomy and excisional biopsy of abdominal lymph node were performed and disease was diagnosed as composite lymphoma , consisting of diffuse large b - cell lymphoma and nodular sclerosing hodgkin lymphoma .
You are an expert at summarizing long articles. Proceed to summarize the following text: although in most cases the cause of chest pain is known to be a benign condition , it is a challenge for general practitioners to identify an acutely dangerous cause , such as pulmonary embolism or acute coronary syndrome , but , on the other hand , to avoid unnecessary testing , referrals , and hospital admissions . a large amount of literature addresses the topic , but the majority focuses on underlying specific conditions or single diseases , such as chronic heart disease , or the data collection is conducted in settings other than general practice . this study was planned to characterize the consultation rate , the interventions , the results of the encounter ( diagnoses ) , and the significantly more frequent accompanying symptoms and associated results of the encounter of patients attending a typical german general practice setting . the registration of the contacts for chest pain was a minor part of the schsische epidemiologische studie in der allgemeinmedizin 2 ( sesam 2 ) study . data from the dutch transition project , a study with one of the largest patient samples in primary care , were also analyzed in detail with regard to chest pain as the reason for encounter . in the course of the sesam 2 study , the saxon society of general medicine ( sgam ) contacted all general practitioners in saxony by mail . the study was set out to document reasons for consultation , diagnostic , and therapeutic interventions as well as the result of consultation ( chosen diagnosis ) . of the 2510 physicians contacted , 270 general practitioners agreed to participate , and 209 cooperated during the complete period of the study ( 1 year ) . cross - sectional data were collected from october 01 , 1999 to september 30 , 2000 . case recording was carried out on 1 day / week ( monday to friday ; either morning or afternoon consultation hours ) , randomly chosen . data were collected for one out of ten patients of every practitioner ( exactly every 10 patient attending the consultation hours ) . it was developed by general practitioners ( leipzig medical school and sgam ) , tested , and evaluated during a pilot trial ( saxon epidemiological study in general practice - sesam 1 ) . each patient 's reasons for encounter , accompanying symptoms , diagnostic interventions , and results of the encounter were documented , as well as therapeutic interventions . as far as possible , data were documented verbatim ( according to the study instructions ) : either as told by the patients ( e.g. , reasons for the encounter ) or in the words of the physician ( e.g. , chronic problems , disease labels , results of encounter or diagnoses ) . only fully completed forms were considered . as described elsewhere , the sesam 2 study provides independent and unbiased cross - sectional data from a typical primary care setting . because all reasons for consulting were investigated and documented , there is no bias toward the investigated reason of encounter . the 1987 version of the international classification of primary care ( icpc ) was used to code the reasons for the consultation . it followed the guidelines of the ethics committee of the saxon chamber of physicians who approved the design and the study . since the data reported by the general practitioners did not allow the identification of single patients and since the study was unlikely to interfere with the treatment of patients or their physical or psychical integrity there was , according to the ( model ) professional code of physicians in germany , no further ethical approval was necessary . publicly available data from the dutch transition project ( described by lamberts and okkes ) were analyzed ( dtp ; total estimation of patients from about 20 dutch general practitioners ; 19852003 ) . because chest pain is not represented by a single icpc code , the codes k01 ( pain attributed to heart ) , k02 ( pressure / tightness attributed to heart ) , k29 ( other symptoms heart / circulatory system ) , l04 ( chest symptoms / complaints ) , r01 ( pain attributed to respiratory system ) , and have been summarized as chest pain . statistical analyses of the data were performed using statistical packages for social sciences ( spss version 15.0 ; spss inc . , in the course of the sesam 2 study , the saxon society of general medicine ( sgam ) contacted all general practitioners in saxony by mail . the study was set out to document reasons for consultation , diagnostic , and therapeutic interventions as well as the result of consultation ( chosen diagnosis ) . of the 2510 physicians contacted , 270 general practitioners agreed to participate , and 209 cooperated during the complete period of the study ( 1 year ) . cross - sectional data were collected from october 01 , 1999 to september 30 , 2000 . case recording was carried out on 1 day / week ( monday to friday ; either morning or afternoon consultation hours ) , randomly chosen . data were collected for one out of ten patients of every practitioner ( exactly every 10 patient attending the consultation hours ) . it was developed by general practitioners ( leipzig medical school and sgam ) , tested , and evaluated during a pilot trial ( saxon epidemiological study in general practice - sesam 1 ) . each patient 's reasons for encounter , accompanying symptoms , diagnostic interventions , and results of the encounter were documented , as well as therapeutic interventions . as far as possible , data were documented verbatim ( according to the study instructions ) : either as told by the patients ( e.g. , reasons for the encounter ) or in the words of the physician ( e.g. , chronic problems , disease labels , results of encounter or diagnoses ) . only fully completed forms were considered . as described elsewhere , the sesam 2 study provides independent and unbiased cross - sectional data from a typical primary care setting . because all reasons for consulting were investigated and documented , there is no bias toward the investigated reason of encounter . the 1987 version of the international classification of primary care ( icpc ) was used to code the reasons for the consultation . it followed the guidelines of the ethics committee of the saxon chamber of physicians who approved the design and the study . since the data reported by the general practitioners did not allow the identification of single patients and since the study was unlikely to interfere with the treatment of patients or their physical or psychical integrity there was , according to the ( model ) professional code of physicians in germany , no further ethical approval was necessary . publicly available data from the dutch transition project ( described by lamberts and okkes ) were analyzed ( dtp ; total estimation of patients from about 20 dutch general practitioners ; 19852003 ) . because chest pain is not represented by a single icpc code , the codes k01 ( pain attributed to heart ) , k02 ( pressure / tightness attributed to heart ) , k29 ( other symptoms heart / circulatory system ) , l04 ( chest symptoms / complaints ) , r01 ( pain attributed to respiratory system ) , and have been summarized as chest pain . statistical analyses of the data were performed using statistical packages for social sciences ( spss version 15.0 ; spss inc . , chicago , usa ) . a total of 8877 consultations were documented in the sesam 2 study and 13,632 reasons for encounter were coded . between 1999 and 2000 , the number of cases reported from each general practitioner 's office ranged from 23 to 54 . there were 5050 ( 56.9% ) female and 3824 ( 43.1% ) male patients reported by 209 general practitioners ; gender was not reported in three cases . age ranged from 2 to 102 years ( mean 51.2 years , standard deviation 20.9 , median 55 years ) . this equates to a frequency of 3.0% [ table 1 for age distribution ] . at 39.3% , the age group 4564 years accounted for the largest number of patients encountering chest pain . of these , 152 ( 56.3% ) were female , and 118 ( 43.7% ) were male . the age - distribution of chest pain patients ( ad ) and the frequency of chest pain patients among all patients in percent of the respective age group ( cp ) in the german sesam 2 study ( total n=8877 ) and the dtp ( total n=149,238 ) more than 50 different symptoms that accompanied chest pain were recorded . a few were statistically significantly associated with chest pain in the sesam 2 study : dyspnea ( 4.8% vs. 0.9% , p < 0.001 ) , accidents / injuries ( 0.4% vs. 0.1% , p = 0.001 ) , and palpitations ( 0.7% vs. 0.3% , p < 0.001 ) . anxiety not other specified ( 3.0% vs. 0.8% ) , fear of a heart disease or respiratory disease were also significantly associated ( 0.7% vs. 0.1% and 0.7% vs. 0.0% , p < 0.05 for each ) with chest pain . nearly , all patients were physically examined ( 92.6% ) . table 2 summarizes the management of chest pain : referrals were made for 11.1% and hospitalization for 4.1% of patients with chest pain . the most frequent medical treatments were follow - up consultation ( 75.6% ) and drug prescription ( 67.4% ) . physician 's action ( % ) in the german sesam 2 study and the dtp concerning the consultations for chest pain results of encounters ( diagnoses ) are summarized in table 3 . thirteen results of encounter occurred significantly more often in patients consulting for chest pain . comparing the number ( n ) and percentage ( % ) of results of encounter ( diagnoses ) in general practice patients with chest pain to those without chest pain ( sesam 2 study ) a total of 149,238 patients were examined for the transition project from 1985 to 2003 ; 84,285 ( 56.5% ) of them were female . in all , 8117 patients suffered from chest pain ; 4333 ( 53.4% ) were female patients . the frequencies were 39.1 for male and 41.5 for female patients per 1000 patient years . among the total number of 597,312 encounters , there were 7220 ( 72.0% ) consultations for new chest pain and 2814 ( 28.0% ) consultations for previously known chest pain , respectively . patients consulting for chest pain also suffered from a cough ( 3.3% ) , dyspnea ( 3.0% ) , and feared diseases of the circulatory system ( 2.8% ) . furthermore , palpitations ( 1.2% ) , general weakness ( 1.1% ) , arm complaints ( 0.7% ) , accidents / injuries ( 0.7% ) , shoulder complaints ( 0.7% ) , back complaints ( 0.7% ) , fever ( 0.6% ) , and fear of heart attack ( 0.6% ) were also common accompanying reasons for encounter in patients consulting for chest pain . the recorded interventions for chest pain patients are summarized in table 2 . in the dutch transition project , general practitioners attributed chest pain most commonly to ischemic heart diseases , pulmonary diseases , and diseases of the musculoskeletal system [ table 3 ] . possible life - threatening dangerous causes in 10,034 consultations with chest pain were : pneumonia ( 0.8% ) , pulmonary embolism ( 0.2% ) , paroxysmal tachycardia ( 0.2% ) and duodenal ulcer , other peptic ulcers , stroke / cerebrovascular insult , ruptured spleen , or toxemia ( pre ) eclampsia , with each accounting for < 0.1% . acute coronary syndrome , acute myocardial infarction , and unstable angina pectoris were classified as ischemic heart disease ( 10.0% ) ; therefore , no explicit frequencies of these dangerous causes of chest pain can be provided . other results of encounters that may have required further investigation included general heart failure and heart valve diseases , cardiac arrhythmias , and suspected ( malignant ) neoplasm . a total of 8877 consultations were documented in the sesam 2 study and 13,632 reasons for encounter were coded . between 1999 and 2000 , the number of cases reported from each general practitioner 's office ranged from 23 to 54 . there were 5050 ( 56.9% ) female and 3824 ( 43.1% ) male patients reported by 209 general practitioners ; gender was not reported in three cases . age ranged from 2 to 102 years ( mean 51.2 years , standard deviation 20.9 , median 55 years ) . this equates to a frequency of 3.0% [ table 1 for age distribution ] . at 39.3% , the age group 4564 years accounted for the largest number of patients encountering chest pain . of these , 152 ( 56.3% ) were female , and 118 ( 43.7% ) were male . the age - distribution of chest pain patients ( ad ) and the frequency of chest pain patients among all patients in percent of the respective age group ( cp ) in the german sesam 2 study ( total n=8877 ) and the dtp ( total n=149,238 ) more than 50 different symptoms that accompanied chest pain were recorded . a few were statistically significantly associated with chest pain in the sesam 2 study : dyspnea ( 4.8% vs. 0.9% , p < 0.001 ) , accidents / injuries ( 0.4% vs. 0.1% , p = 0.001 ) , and palpitations ( 0.7% vs. 0.3% , p < 0.001 ) . anxiety not other specified ( 3.0% vs. 0.8% ) , fear of a heart disease or respiratory disease were also significantly associated ( 0.7% vs. 0.1% and 0.7% vs. 0.0% , p < 0.05 for each ) with chest pain . nearly , all patients were physically examined ( 92.6% ) . table 2 summarizes the management of chest pain : referrals were made for 11.1% and hospitalization for 4.1% of patients with chest pain . the most frequent medical treatments were follow - up consultation ( 75.6% ) and drug prescription ( 67.4% ) . physician 's action ( % ) in the german sesam 2 study and the dtp concerning the consultations for chest pain results of encounters ( diagnoses ) are summarized in table 3 . thirteen results of encounter occurred significantly more often in patients consulting for chest pain . comparing the number ( n ) and percentage ( % ) of results of encounter ( diagnoses ) in general practice patients with chest pain to those without chest pain ( sesam 2 study ) a total of 149,238 patients were examined for the transition project from 1985 to 2003 ; 84,285 ( 56.5% ) of them were female . in all , 8117 patients suffered from chest pain ; 4333 ( 53.4% ) were female patients . the frequencies were 39.1 for male and 41.5 for female patients per 1000 patient years . among the total number of 597,312 encounters , there were 7220 ( 72.0% ) consultations for new chest pain and 2814 ( 28.0% ) consultations for previously known chest pain , respectively . patients consulting for chest pain also suffered from a cough ( 3.3% ) , dyspnea ( 3.0% ) , and feared diseases of the circulatory system ( 2.8% ) . furthermore , palpitations ( 1.2% ) , general weakness ( 1.1% ) , arm complaints ( 0.7% ) , accidents / injuries ( 0.7% ) , shoulder complaints ( 0.7% ) , back complaints ( 0.7% ) , fever ( 0.6% ) , and fear of heart attack ( 0.6% ) were also common accompanying reasons for encounter in patients consulting for chest pain . the recorded interventions for chest pain patients are summarized in table 2 . in the dutch transition project , general practitioners attributed chest pain most commonly to ischemic heart diseases , pulmonary diseases , and diseases of the musculoskeletal system [ table 3 ] . possible life - threatening dangerous causes in 10,034 consultations with chest pain were : pneumonia ( 0.8% ) , pulmonary embolism ( 0.2% ) , paroxysmal tachycardia ( 0.2% ) and duodenal ulcer , other peptic ulcers , stroke / cerebrovascular insult , ruptured spleen , or toxemia ( pre ) eclampsia , with each accounting for < 0.1% . acute coronary syndrome , acute myocardial infarction , and unstable angina pectoris were classified as ischemic heart disease ( 10.0% ) ; therefore , no explicit frequencies of these dangerous causes of chest pain can be provided . other results of encounters that may have required further investigation included general heart failure and heart valve diseases , cardiac arrhythmias , and suspected ( malignant ) neoplasm . in a daily general practice setting , we found the consultation rate of chest pain ranging from 0.7% to 3.0% . in the german sesam 2 study and the dutch transition project , dyspnea , anxiety , fear of diseases , and injuries the physician 's advice , drug prescription , and follow - up consultation were the most frequent therapeutic interventions . chest pain was most commonly attributed to cardiovascular diseases , problems of the musculoskeletal , or respiratory system . acutely dangerous courses were rare . the consultation rate of chest pain as a reason for the encounter was remarkably higher in patients older than 45 years . this can be explained by a rising prevalence of chronic problems ( e.g. , musculoskeletal or cardiovascular ones ) and by the increased rate of healthcare seeking in patients aged 60 years and over and by the ascending frequency of cardiovascular check - ups in these patients . other studies reported chest pain frequencies of about 1.5% ( sweden ) , 0.7% ( iceland ) , and 15.5/1000 patient - years ( great britain ) but similar findings especially the nearly doubling of the chest pain frequency from younger adults to the elderly , as seen in the dutch transition project but also a first peak in the age group of 2140 years . the variation in the reported consultation frequencies [ table 4 ] may be explained by the different geographic backgrounds and different sampling methods of the mentioned studies . furthermore , there is an influence on how individual patients of different cultural backgrounds respond to chest pain , for example to symptoms of the acute coronary syndrome . these assumptions are supported by the fact that the swiss thoracic pain in community ( topic ) study , which was performed in a time period and with a population comparable to those in the german sesam 2 study , revealed a nearly equal consultation frequency of chest pain [ table 4 ] . however , the higher rate in the german sesam 2 study is unlikely to be influenced by attention bias since the study estimated total morbidity . the frequency of chest pain ( patients that consulted for chest pain related to all included patients of the respective study ) and the results of encounter in different general practice studies . the data and comparison points were adopted from verdon et al . and supplemented with the results of the german sesam 2 study and those of the dtp there are noticeable differences in diagnostic and therapeutic interventions between the german sesam 2 study and the dutch transition project [ table 2 ] . regarding the results of encounter in the german sesam 2 study and other investigations , we can conclude that some of the diagnostic interventions may have been not necessary . regarding other investigations of chest pain in general practice the design of the swiss , topic study was most similar to that of the german sesam 2 study . the percentages of the diagnostic and therapeutic interventions were comparable to that of the german sesam 2 study . this is the case because the general practitioners documented the individual case by their own . this limitation could have been excluded by a total estimation of all consulting patients and electronic patient records . however , the electronic patient recording does not necessarily imply a high quality of documentation . the results of our own investigation , as well as the findings reported by others , can not provide information on the use of established diagnostic scores or applied diagnostic rapid - tests . the results of encounter from patients that reported chest pain are presented in table 4 , the underlying categories , which could be partially critically discussed , were adopted from verdon et al . despite varying percentages of different organ systems being identified as the reason of chest pain , in five of the seven assessed studies , musculoskeletal problems were the most frequent underlying cause of chest pain in general practice . found cardiovascular problems ranked second in four of seven studies and ranked first only in the north - american ambulatory sentinel practice network ( aspn ) study and the dutch transition project . the most rare results of encounter belong to gastrointestinal diseases . in the dutch transition project , this is generally in accordance with the results of others that showed that only 10% of the results of encounter in general practice can be assigned to a reliable diagnosis , but up to 50% are classified as symptoms and 40% as named syndromes . despite these limitations , it has to be pointed out that acutely dangerous courses such as suspected pulmonary embolism and suspected myocardial infarction occurred in 14% of all patients that encountered for chest pain . however , only in the north - american aspn and the german sesam 2 study suspected myocardial infarction was documented regularly frequently ( more than one case in a total of 3000 consultations ) . suspected pulmonary embolism was documented regularly frequently only in the german sesam 2 study . as described elsewhere , the german sesam 2 study supplies independent and representative cross - sectional data from a daily general practice setting . a total of 209 general practitioners cooperated during the complete period of the study this is a much higher number than in the studies mentioned by verdon et al . in the dutch transition project , since both studies estimated total morbidity , there is no attention or selection bias toward chest pain . the german sesam 2 study is regionally specific ; only general practitioners of saxony were contacted and participated . house calls and out - of - hour contacts were not estimated in the study . an estimation of all patients was not possible , and the data do not represent the episodes of care . a valid statement on the quality of diagnoses of the german sesam 2 study of the total number of general practitioners contacted , 10.9% participated in the german sesam 2 study . however , the other studies [ table 4 ] were conducted with a much smaller number of general practitioners . this makes them more sensitive to effects of bias through specifics of individual patient populations and general practitioners . for this reason , there is no significant limitation resulting from a participation rate of 209 general practitioners in the german sesam 2 study . the dutch transition project has a large patient sample but was acquired from a small number of general practitioners . this is due to methodological differences and different regions and time periods of data assessment . however , during the time of data assessment until publication there have been no fundamental changes beside an increasing mean age in the study population or in the german general practices , so it can be assumed that the spectrum of reasons for encounter and the approaches toward it changed only slightly . since there was no other , more recently , assessed data published our results meanwhile do broaden the current knowledge on the topic . as described elsewhere , the german sesam 2 study supplies independent and representative cross - sectional data from a daily general practice setting . a total of 209 general practitioners cooperated during the complete period of the study this is a much higher number than in the studies mentioned by verdon et al . in the dutch transition project , since both studies estimated total morbidity , there is no attention or selection bias toward chest pain . the german sesam 2 study is regionally specific ; only general practitioners of saxony were contacted and participated . house calls and out - of - hour contacts were not estimated in the study . an estimation of all patients was not possible , and the data do not represent the episodes of care . a valid statement on the quality of diagnoses of the german sesam 2 study of the total number of general practitioners contacted , 10.9% participated in the german sesam 2 study . however , the other studies [ table 4 ] were conducted with a much smaller number of general practitioners . this makes them more sensitive to effects of bias through specifics of individual patient populations and general practitioners . for this reason , there is no significant limitation resulting from a participation rate of 209 general practitioners in the german sesam 2 study . the dutch transition project has a large patient sample but was acquired from a small number of general practitioners . this is due to methodological differences and different regions and time periods of data assessment . however , during the time of data assessment until publication there have been no fundamental changes beside an increasing mean age in the study population or in the german general practices , so it can be assumed that the spectrum of reasons for encounter and the approaches toward it changed only slightly . since there was no other , more recently , assessed data published our results meanwhile do broaden the current knowledge on the topic . chest pain occurs mainly due to musculoskeletal , circulatory , psychogenic , or digestive problems . in a daily general practice setting the publication was supported from the german research foundation ( dfg ) and university of leipzig within the program of open access publishing . the publication was supported from the german research foundation ( dfg ) and university of leipzig within the program of open access publishing .
objective : chest pain is a common reason for an encounter in general practice . the present investigation was set out to characterize the consultation rate of chest pain , accompanying symptoms , frequency of diagnostic and therapeutic interventions , and results of the encounter.materials and methods : cross - sectional data were collected from randomly selected patients in the german schsische epidemiologische studie in der allgemeinmedizin 2 ( sesam 2 ) and analyzed from the dutch transition project.results:overall , 270 patients from the sesam 2 study consulted a general practitioner due to chest pain ( 3% of all consultations ) . chest pain was more frequent in people aged over 45 years . the most common diagnostic interventions were physical examination , electrocardiogram at rest and analysis of blood parameters . for the majority of cases , the physicians arranged a follow - up consultation or prescribed drugs . the transition project documented 8117 patients reporting chest pain with a frequency of 44.5/1000 patient years ( 1.7% of all consultations ) . physical examination was also the most common diagnostic intervention , and physician 's advice the most relevant therapeutic one.conclusion:the most common causes for chest pain were musculoskeletal problems followed by cardiovascular diseases . ischemic heart disease , psychogenic problems , and respiratory diseases each account for about 10% of the cases . however , acutely dangerous causes are rare in general practice .
You are an expert at summarizing long articles. Proceed to summarize the following text: merely looking at a stranger s twitter or facebook feed is nt interesting , because it seems like blather . follow it for a day , though , and it begins to feel like a short story ; follow it for a month , and it s a novel.thompson ( 2008 ) merely looking at a stranger s twitter or facebook feed is nt interesting , because it seems like blather . follow it for a day , though , and it begins to feel like a short story ; follow it for a month , and it s a novel . although many social media platforms are mainly used for maintaining existing relationships , it is also common to stumble across the messages of strangers . on twitter for example , it is relatively common to follow people one knows only online ( utz , 2016 ) . a survey among twitter users has shown that people develop ambient intimacy , i.e. a feeling of closeness to others followed on social media , for some of the people they follow on twitter ( lin , levordasha , & utz , 2016 ) . classical studies on relationship formation focus on the role of intimacy in self - disclosure ( altman & taylor , 1973 ) , although studies on ( semi- ) public social media have shown that entertaining posts can also create a feeling of closeness ( lin et al . however , as these studies have been correlational and relied on self - reported judgments / recall of the content of posts , it is not clear which factors drive the development of a feeling of closeness . to answer this question , we conducted two experiments in which we varied the number of posts and manipulated for the target person not only the intimacy but also the narrativity of self - disclosure . the latter factor has hitherto been neglected , although the quote at the beginning of this paper indicates that narrativity and story value of posts might matter . additionally , we look at potential influence factors such as perceived appropriateness and entertainment value of posts . by experimentally disentangling the role of intimacy and narrativity , we contribute to a better understanding of the processes underlying relationship formation on social media . in the following paragraphs , we will introduce the concepts of self - disclosure frequency , intimacy , and narrativity on social media , and its difference to self - disclosure in traditional one - to - one communication . relevant previous research on the effects of self - disclosure on familiarity , closeness , and social attraction will be discussed . conceptually speaking , broadcasting status updates / posts on social media that contain any form of self - information can be treated as self - disclosure ( lin et al . , 2016 ) . the posts could contain descriptive information such as what one has done today , and/or evaluative information such as how one feels about an event . in previous research , the degree of self - disclosure was often assessed along two dimensions : disclosure breadth ( e.g. , the amount of self - relevant statements made during an interaction ) and disclosure depth ( i.e. , the level of disclosure intimacy ) ( altman & taylor , 1973 ) . public self - disclosures on social media have some unique properties compared to self - disclosure in traditional one - to - one communication . first , when the disclosing self - information is highly intimate , it is more likely to be perceived as inappropriate when one broadcasts it online than discloses it privately ( bazarova , 2012 ) ; therefore , intimate self - disclosure on social media often decreases interpersonal attraction ( baruh & cemalclar , 2015 ) . second , public self - disclosure is often not directed to a single person , but simultaneously to several people . disclosure recipients might not feel addressed in this case ; and self - disclosure might have smaller effects on relationships ( burke & kraut , 2014 ) . third , public self - disclosure from a specific person is often embedded in a stream of others information ; hence disclosure recipients may not pay enough attention to it . in addition to the two dimensions of self - disclosure breadth and depth , we assume that self - disclosure narrativity is also important in the context of social media . we will discuss the role of disclosure intimacy and narrativity later , and first introduce the literature on the effects of self - disclosure frequency on familiarity , closeness , and social attraction . we chose these three dependent variables because they were often discussed in previous literature ( norton et al . 2012 ) , but conceptually they are slightly different from each other . in this paper , familiarity refers to the state of being accustomed to something or someone . it is knowledge of something or someone , which mainly depends on disclosure frequency ( finkel et al . , 2015 , pp . 139 ) , regardless of the valence . closeness and social attraction are more likely to be influenced by the content of self - disclosure . closeness covers the emotional facet , whereas social attraction refers to the behavioral component , e.g. wanting to have a coffee with the target . even though there are some differences in private and public self - disclosure , recent studies indicated that browsing social media helps to enhance familiarity , creates awareness / knowledge of online contacts ( levordashka & utz , 2016 ) , and generates a feeling of closeness ( lin et al . , 2016 ) . familiarity is generated when an individual has a certain level of exposure to the target person ( finkel et al . therefore , a greater amount of public self - disclosure on social media should also generate a higher level of familiarity . when it comes to closeness and attraction , findings are less consistent . in private conversations , when perceived as appropriate , greater amounts of disclosure information were often associated with more liking of the discloser ( for review , see collins & miller , 1994 ) . for unacquainted strangers , self - disclosure often leads to more closeness ( a. aron , melinat , aron , vallone , & bator , 1997 ) , which creates familiarity - based liking ( berger and calabrese , 1975 , zajonc , 1968 ) , and positive interpersonal impressions such as social attraction ( sprecher et al . , 2012 ) . this is due to mere exposure effect and uncertainty reduction theory . the former asserted that the more frequently one is exposed to a certain thing or person , the more likable that thing or person appears to be ( moreland and zajonc , 1982 , zajonc , 1968 ) . the latter theory indicated that the more one is exposed to other s self - disclosure , the more uncertainty is reduced , therefore liking is increased ( sunnafrank , 1986 ) . however , other researchers have found that more information about a person may decrease liking due to a higher level of perceived dissimilarity . less is more hypothesis ( norton , frost , & ariely , 2007 ) . the mixed findings can be explained by the information - processing approach of attraction ( ajzen , 1977 , dalto et al . , 1979 ) . it suggests that liking is determined by having positive beliefs about an individual : the more positive the beliefs , the greater the attraction ; however , if the content of self - discloser leads to negative beliefs ( e.g. , due to perceived dissimilarity or inappropriateness ) , the attraction should be decreased ( ajzen , 1977 ) . similar to the familiarity and attraction link , we assume that , after reading other s public self - disclosure on social media , a feeling of closeness can be generated only under certain conditions ( i.e. , when positive beliefs are generated ) . in this case , in addition to disclosure frequency , the content of self - disclosure is more important in predicting social attraction . in offline relationship building , but it is still a debatable question whether disclosing intimate information promotes or undermines interpersonal attraction and closeness on sns . for existing interpersonal relationships , researchers have found that receiving a larger proportion of superficial disclosures decreases relationship satisfaction ( rains , brunner , & oman , 2014 ) . self - disclosure on sns , when perceived as more intimate , increases the feeling of connection toward existing online friends ( lin et al . , 2016 , utz , 2015 ) . however , such a positive effect of disclosure intimacy on building interpersonal relationships is stronger for messages on private channels than for public status updates ( bazarova , 2012 ) . in the context of public status updates , we focus on situations in which an individual had no previous interactions with a target , using a so - called zero - acquaintance paradigm in which perceivers make judgments about strangers without having the opportunity to interact ( albright , kenny , & malloy , 1988 ) . baruh and cemalclar ( 2015 ) have found that broadcasting intimate disclosure messages may attract more attention but not necessarily increase attraction . however , it is important to take the role of appropriateness into account , as intimate disclosure in public is often perceived as inappropriate ( bazarova , 2012 ) . in baruh and cemalclar s ( 2015 ) study , the level of disclosure appropriateness was altered essentially when the level of disclosure intimacy was manipulated . it was difficult to conclude whether the decreased attraction is because of the high - intimacy or high - inappropriateness . the current study aims to examine the role of disclosure intimacy by minimizing the difference in the perceived appropriateness of high- and low - intimacy disclosure messages . we used stimulus material that was judged as appropriate in a pretest , and expected that under these circumstances intimate self - disclosure should have a positive role in increasing closeness and social attraction . a second factor that has been neglected in prior research on the development of closeness on social media we conceptualized narrative self - disclosure as revealing information about oneself in a story - telling way . on social media , pieces of self - disclosure information are treated as high in narrativity when they , taken together , are able to form a continuous and coherent story . vorderer ( 2016 ) argued that narratives play a central role in new media . for example , people are transported into the narratives in novels or movies ( bilandzic , 2006 , green et al . , 2004 ) , and they may develop a feeling of closeness to the character by merging into the narrative . compared with the same events that are portrayed through less narrativity , higher levels of narrativity often correspond to greater vividness , ease of narrative understanding , engagement , and emotional responses ( busselle & bilandzic , 2009 ) . considering the strong desire of people for stories , we thus assume that narrative self - disclosure should have a positive effect on perceived closeness and social attraction . in sum , we developed the following hypotheses.h1the amount of public self - disclosure increases familiarity.h2intimate self - disclosure , when perceived as appropriate , increases ( a ) perceived closeness and ( b ) social attraction.h3narrative self - disclosure increases ( a ) perceived closeness and ( b ) social attraction . the amount of public self - disclosure increases familiarity . intimate self - disclosure , when perceived as appropriate , increases ( a ) perceived closeness and ( b ) social attraction . narrative self - disclosure increases ( a ) perceived closeness and ( b ) social attraction . in order to examine these hypotheses , two pretests and two studies were conducted . the two pretests were conducted in order to create proper stimuli before conducting the main studies . the initial main study was followed up with an online version of the same study : study 1 was conducted in a lab and with a limited amount of participants ; study 2 aimed to replicate the results of study 1 , and was an online questionnaire with a larger sample size . in order to examine h1 , a special design that enables a quasi - experience of sns browsing was applied ( see a sample of the stimuli in fig . 1 ) . we created a fake sns called social net , and mocked - up web pages that resemble the layout of facebook news feed , including posts , user names , and profile pictures . in our scenario , the target discloser posted 5 times , and the other four disclosers posted 5 times , 3 times , 1 time , and 1 time , respectively . in order to test h2 and h3 , we adopted a 2 ( high- vs. low - intimacy disclosure ) 2 ( high- vs. low - narrativity disclosure ) between - subject design . we created four versions of disclosure messages for the target discloser . in each condition , the five manipulated disclosure posts of a target discloser ( as described in appendix a ) were embedded ( in the same order ) in another ten distracting posts from the other four disclosers / distractors ( see fig . 1 ) . two pretests were conducted in order to compose the stimuli for the main experiment ( see in appendix a ) . in the first pretest , 56 disclosure messages were selected and adapted from real tweets on twitter . sixty - four german participants evaluated the levels of intimacy and appropriateness for each individual message after translation into german . based on these results , we composed four versions of target posts ( i.e. , disclosure messages of the target person ) after adjusting the level of narrativity . this version of posts was similar to the final version of posts in appendix a. we manipulated the level of intimacy by adding information about one s emotions , inner thoughts , and beliefs ; and we manipulated the level of narrativity by adding more details and transitional descriptions . the levels of disclosure narrativity , intimacy , and appropriateness of each stimulus were assessed by another 52 german participants using a between - subject design . the results of the second pretest indicated that such stimuli can successfully manipulate the levels of disclosure intimacy and narrativity accordingly , and the levels of appropriateness were above the middle point of 4 in all conditions . in study 1 , participants were asked to complete an online questionnaire in the lab . they were asked to imagine that they just created an account on a newly launched sns called social net and had followed some other users profile as recommended by the system . they were also informed that many social net users feel at ease to keep their postings as a digital public diary on that sns . this instruction helped us to investigate the effect of self - disclosure intimacy when public self - disclosure is legitimized and more likely to be perceived as appropriate . it hopefully can minimize the difference of the appropriateness level between high- and low - intimacy conditions . then participants were randomly assigned into one of the four conditions , and they were allowed to browse the stimuli as long as they wished to . as soon as they finished browsing , the levels of familiarity were measured by asking ten profiles were shown in the first column of the question matrix ( in a randomized order ) : the profiles of the five disclosers that were shown in the stimulus and five new profiles that served as distractors . participants could choose do not remember if they felt unfamiliar with the profile owner , and they were supposed to choose that option for the distractors . first , the five profile pictures of the disclosers were shown again with the following instruction : compared to other users on social net , how close do you feel to the following profile owners . participants could score between 1 and 7 , or choose no impression if they could not recall . second , immediately after the one - item measure of closeness , the perceived closeness to the target discloser was measured by using the propinquity scale ( walther & bazarova , 2008 ) with a 7-point semantic differential scale ( see more information about measurements in appendix b ) . in addition , perceived social attraction of the target was measured by social attraction scale with a 7-point likert scale ( mccroskey & mccain , 1974 ) . then the five posts of the target discloser ( target posts ) were shown again , and participants were asked to evaluate the posts of the target discloser with several seven - point semantic differential scales . the perceived levels of intimacy and appropriateness for the message sets were measured with scales used in bazarova s ( 2012 ) study . disclosure narrativity of the target posts was measured with three self - created items about disclosure coherence ( e.g. , not coherent - coherent ) . previous research found that the entertainment value of messages significantly predicts the feeling of closeness to the discloser on social media ( lin et al . therefore , we also measured the perceived entertainment value of the target posts with one - item for exploratory purposes . a summary of the items for these measurements and the cronbach s alphas are reported in appendix b. other demographical information such as age , gender , as well as social media usage behavior was measured at the end of the questionnaire . participants , in both pretests and the first study , were recruited from a local panel in germany . participants should be a social media user and 18 years and older , and they were paid according to a standard of 8 euro / hour . although we initially aimed for a sample size of 160 , only 145 participants completed the main experiment after we extended the lab study for another three weeks . in order to prevent participants clicking through the stimuli without browsing , we dropped the cases in which participants spent less than 30 s in reading each stimulus . we estimated each status update would take about 2 s to skim through ; therefore 15 posts might take 30 s. this selection criteria resulted in a final sample of 139 ( 29 males ; mage = 23.58 years ; sd = 4.29 years ) . table 1 depicts the descriptive results for the degrees of familiarity with the five existing and the five new profile owners . out of 139 participants , only 5 participants ( 1.4% ) reported do not remember seeing the target profile owner , whereas , for those distracting new profiles , more than 101 participants ( 72.7% ) correctly recognized that they were not shown in the stimuli before . in addition , the level of familiarity with the target discloser ( m = 4.69 , sd = 1.86 ) was already higher than the level of familiarity with the third discloser who posted three times ( m = 4.06 , sd = 1.67 ) , t(124 ) = 4.81 , p < 0.001 , let alone those who disclosed less often ( see more comparison information in table 1 ) . we also measured the feelings of closeness to the five disclosers ( see results in table 2 ) . similar to the pattern of familiarity , participants felt closer to the profiles owners who disclose more frequently : the target profile owner who posted five times ( m = 3.64 , sd = 1.85 ) was perceived as closer compared to the profile owner who posted three times ( m = 3.14 , sd = 1.60 ) , t(129 ) = 3.06 , p = 0.001 , who was in turn perceived as emotionally closer than the two disclosers who posted less frequently ( see more information in table 2 ) . descriptive results for the key variables can be found in table 3 , and correlation statistics can be found in table 4 . the results of the manipulation check showed that we manipulated the disclosure intimacy and narrativity successfully . a two - way anova showed participants in the high - intimacy condition perceived the target posts as more intimate ( m = 5.69 , sd = 0.91 ) than that in the low - intimacy condition ( m = 4.49 , sd = 1.08 ) , f(1 , 135 ) = 49.67 , p < there was no main effect of narrativity condition , nor an interaction effect for perceived intimacy . another two - way anova showed that participants in the high - narrativity condition perceived the target posts as more coherent as a story ( m = 6.02 , sd = 0.96 ) than that in the low - narrativity condition ( m = 3.89 , sd = 1.39 ) , f(1 , 135 ) = 155.61 , p < 0.001 , p = 0.447 . as expected , there was no main effect of intimacy condition , nor an interaction effect for perceived coherence . in previous research , disclosure appropriateness was highly associated with disclosure intimacy on social media ( bazarova , 2012 ) . therefore , we also examined whether there is a group difference of appropriateness using a two - way anova . the result showed that participants in the high - intimacy condition ( m = 4.38 , sd = 1.10 ) also perceived the disclosure as slightly less appropriate than that in the low - intimacy condition ( m = 4.83 , sd = 1.27 ) , f(1 , 135 ) = 4.88 , p = 0.029 , p = 0.035 . this result showed that it is difficult to disentangle the concepts of perceived intimacy and perceived appropriateness in the context of public self - disclosure on social media . even though we tried to make intimate public self - disclosure as appropriate as possible , it was still rated as slightly less appropriate than non - intimate public self - disclosure . most importantly , the mean values of disclosure appropriateness were above four in all conditions , indicating that we succeeded in constructing appropriate posts . therefore , their main effects on perceived closeness and social attraction can still be tested by anova . two - way anovas were applied to examine the main effects of disclosure intimacy and narrativity on perceived closeness ( one - item closeness and propinquity ) and social attraction . for the one - item measure of closeness , the result of two - way anova revealed a significant interaction effect between the intimacy level and the narrativity level , f(1 , 131 ) = 6.69 , p = 0.011 , p = 0.048 . under the condition of low - narrativity , participants in the high - intimacy group ( m = 4.14 , sd = 1.87 ) reported a higher level of perceived closeness to the target discloser than those in the low - intimacy group ( m = 2.50 , sd = 1.38 ) , t ( 65 ) = 3.98 , p < 0.001 , cohen s d = 0.814 ; but under the condition of high - narrativity , there was not a significant difference among high - intimacy ( m = 3.88 , sd = 1.79 ) and low - intimacy ( m = 3.79 , sd = 1.79 ) groups . under the condition of low - intimacy , participants in the high - narrativity group ( m = 3.79 , sd = 1.79 ) reported a higher level of perceived closeness than those in the low - narrativity group ( m = 2.50 , sd = 1.38 ) , t ( 62 ) = 3.21 , p = 0.001 , cohen s d = 0.993 ; but under the condition of high - intimacy , no significant difference among high - narrativity ( m = 4.14 , sd = 1.87 ) and low - narrativity ( m = 3.88 , sd = 1.79 ) groups was found . as expected , there was a positive main effect of intimacy on closeness , f(1 , 131 ) = 8.31 , p = 0.005 , p = 0.059 . interestingly , when we dropped the cases in which participants perceived the target posts as inappropriate ( < 4 ) , the positive main effect of disclosure intimacy on perceived closeness was stronger , f(1 , 95 ) = 13.92 , p < 0.001 , p = 0.128 . these results supported h2a and h3a partially , because we did not expect an interaction effect to be significant . for the propinquity measure , the result of two - way anova yielded only a weak interaction effect , f(1 , 135 ) = 9.82 , p = 0.043 , p = 0.030 . the pattern was similar to the one item measure of closeness : under the condition of low - narrativity , participants in the high - intimacy group ( m = 3.81 , sd = 1.60 ) reported a higher level of propinquity toward the target discloser than those in the low - intimacy group ( m = 3.03 , sd = 1.51 ) , t ( 68 ) = 2.08 , p = 0.021 , cohen s d = 0.498 . but no significant group difference was found among high - intimacy ( m = 3.62 , sd = 1.60 ) and low - intimacy ( m = 3.91 , sd = 1.41 ) groups under the condition of high - narrativity . under the condition of low - intimacy , participants in the high - narrativity group ( m = 3.91 , sd = 1.40 ) reported a higher level of perceived closeness than those in the low - narrativity group ( m = 3.03 , sd = 1.51 ) , t ( 65 ) = 2.47 , p = 0.008 , cohen s d = 0.604 . for social attraction , the results of two - way anova showed a negative main effect of disclosure intimacy , f(1 , 135 ) = 20.29 , p = 0.001 , p = 0.076 . note that when we dropped the cases in which participants perceived the target posts as inappropriate ( < 4 ) , the negative main effect of disclosure intimacy on social attraction disappeared , f(1 , 98 ) = 2.74 , p = 0.101 , p = 0.027 . no further significant effect of narrativity was observed ; therefore h3b was also not supported . utilizing an experimental design , study 1 showed that it is possible to develop a feeling of familiarity and a relatively higher feeling of closeness toward a discloser after browsing his / her posts that were embedded in a stream of other s updates . a higher amount of self - disclosure is beneficial for creating a feeling of familiarity and closeness . the role of disclosure intimacy and narrativity were complex : for closeness , we found an interaction effect , indicating that one factor , either intimacy or narrativity , was sufficient to elicit a feeling of closeness . in addition , disclosure intimacy had a positive main effect on the one - item measure of closeness but had a negative effect on social attraction . the correlational statistics in table 4 also indicated that the correlation between the perceived appropriateness and social attraction was higher than the correlation between perceived appropriateness and closeness . however , when we dropped the cases in which participants rated the target posts as inappropriate ( < 4 ) , the positive effect of disclosure intimacy on closeness turned stronger , and the negative main effect of disclosure intimacy on social attraction disappeared . these results provided evidence for the important role of perceived appropriateness , especially when it comes to social attraction . intimate public self - disclosure is thus a double - edged sword : it may increase a feeling of closeness , but it may also decrease social attraction when it is perceived as inappropriate . however , there are some limitations : first , the sample size was not as large as we aimed for since it was difficult to get enough participants to a lab study during that time period . second , the results were not exactly the same between the two measures of closeness ( the one - item question and the propinquity scale ) . third , the interaction effects of closeness between narrative disclosure and intimate disclosure were unexpected . study 2 was an online study in order to collect a larger and more heterogeneous sample of participants . the design of study 2 , including measures and the sequence of measures , was similar to the study 1 . the reliability of scales can be found in appendix b. we were concerned that participants might be less devoted compared to the participants in a lab study . therefore , the stimuli were displayed a second time after the closeness and familiarity measures . participants can then have a second chance to look at the stimuli if they paid less attention to it the first time . based on the study 1 , we intended to recruit at least 400 participants for study 2 . we recruited participants online , including sending invitation emails to several german university email lists and posting the questionnaire links on facebook and some forums . participants who completed this online study were entered into a raffle to receive a 10 euro amazon voucher , the chance of winning was one out of each five participants . similar to the selection criteria in study 1 , participants who spent less than 30 s in reading each stimulus were dropped . this resulted in a final sample of 433 participants ( 161 males ; mage = 25.82 years ; sd = 6.38 years ) . table 5 depicts the descriptive results for the degrees of familiarity with five existing and five new profile owners . among 433 participants , 39 participants ( 9.0% ) reported do not remember when seeing the target profile owner , whereas , for each distractor s profile , at least 292 participants ( > 67.4% ) correctly recognized that this distractor was not shown before . the level of familiarity with the target discloser ( m = 4.61 , sd = 1.79 ) was already higher than the level of familiarity with the third discloser who posted three times ( m = 3.84 , sd = 1.70 ) , t(317 ) = 7.69 , p < 0.001 , supporting h1 ( see more information in table 5 ) . these results were similar to that in study 1 , but the error rate was higher compared to study 1 , which indicates that the online study has more random noises . similar to the results in study 1 , participants in study 2 also indicated that they felt relatively closer to disclosers who disclose more frequently ( see results in table 6 ) . the target discloser who posted five times ( m = 3.42 , sd = 1.80 ) was perceived as emotionally closer compared to the discloser who posted three times ( m = 3.14 , sd = 1.61 ) , t(337 ) = 2.65 , p = 0.004 , who was in turn perceived as emotionally closer than the two disclosers who posted less frequently . the descriptive results and correlation statistics can be found in table 3 , table 4 respectively . a two - way anova for perceived intimacy indicated a significant main effect for both manipulated level of intimacy , f(1 , 429 ) = 56.53 , p < 0.001 , p = 0.116 , and manipulated level of narrativity , f(1 , 429 ) = 11.89 , p < 0.001 , p = 0.027 , and no significant interaction effect was observed here . a two - way anova for perceived coherence showed a significant main effect of manipulated narrativity , f(1 , 429 ) = 310.61 , p < 0.001 , p = 0.420 , and there were no significant main effect of manipulated intimacy and interaction effect . as expected , the results of manipulation check showed that participants in the high - intimacy condition perceived the target posts as more intimate ( m = 5.28 , sd = 1.26 ) than that in the low - intimacy condition ( m = 4.38 , sd = 1.25 ) , t(431 ) = 7.36 , p < 0.001 , cohen s d = 0.708 ; and participants in the high - narrativity condition perceived the target posts as more coherent ( m = 5.98 , sd = 1.12 ) than that in the low - narrativity condition ( m = 3.79 , sd = 1.46 ) , t(431 ) = 17.63 , p < 0.001 , cohen s d = 1.687 . unfortunately , participants in the high - narrativity condition also perceived the target posts as slightly more intimate ( m = 5.02 , sd = 1.28 ) than participants in the low - narrativity condition ( m = 4.62 , sd = 1.37 ) , t(431 ) = 3.10 , p = 0.001 , cohen s d = 0.298 . another two - way anova showed that perceived appropriateness also differed across conditions : participants in the high - intimacy condition ( m = 4.44 , sd = 1.17 ) perceived the target posts as less appropriate than that in the low - intimacy condition ( m = 4.96 , sd = 1.16 ) , f(1 , 429 ) = 21.62 , p < 0.001 , p = 0.048 ; participants in the high - narrativity condition ( m = 4.83 , sd = 1.19 ) perceived the target posts as more appropriate than that in the low - narrativity condition ( m = 4.55 , sd = 1.17 ) , f(1 , 429 ) = 6.21 , p = 0.013 , p = 0.014 . however , values were significantly above 4 in all conditions , indicating that in general the posts were perceived as appropriate . we will nevertheless additionally run some exploratory linear regressions due to the confounded nature of the data after reporting the anova results for closeness and social attraction . for the two measures of closeness , the normality assumptions were not met , and for social attraction , the assumption of homogeneity of variance was not met . therefore , bootstrapping strategies with 5000 repetitions were used here : two - way anovas ( manipulated intimacy manipulated narrativity ) were conducted for the three measures of closeness and social attraction . for both , the one - item measure of closeness and propinquity , however , there were marginal positive effects of manipulated narrativity on closeness [ 95% ci : 0.020.93 ; 90% ci : 0.050.86 ] and propinquity [ 95% ci : 0.010.75 ; 90% ci : 0.050.87 ] . for social attraction , the 95% confidence interval for manipulated intimacy contained zero [ 0.590.13 ] , but manipulated narrativity was above zero [ 0.080.73 ] . that means participants in the high - narrativity condition ( m = 4.61 , sd = 1.29 ) rated the target discloser as more socially attractive than participants in the low - narrativity condition ( m = 4.25 , sd = 1.32 ) , t(431 ) = 2.80 , p = 0.003 . but the effect of manipulated narrativity on social attraction was no longer significant [ 95% ci : 0.110.56 ] after we excluded the cases in which target posts were perceived as inappropriate ( < 4 ) . in study 2 , h2b was rejected , and h3b was partially supported . because the manipulated narrativity changed the level of perceived intimacy in study 2 , it is unclear whether the narrativity effect is ( also ) due to intimacy . hence , measured values of perceived intimacy and narrativity should be used for data analysis . in addition , we later realized that the manipulation of narrativity also changed the level of perceived entertainment value ( in addition to perceived appropriateness ) . therefore , stepwise regressions were used : for each dependent variable , the perceived intimacy and coherence were first added into the linear regression model ( step 1 ) , and then the potentially confounding / mediating variables such as perceived appropriateness and entertainment value were added as predictors ( step 2 ) . the assumption of independent errors was not always met for all linear regressions ; hence we used bootstrapping strategies with 5000 repetitions and 95% confidence intervals . the results can be found in table 7 ( for study 1 ) and table 8 ( for study 2 ) . for the results of step 1 , perceived intimacy was only positively associated with the one - item closeness measure in study 1 , and perceived coherence had a positive effect on propinquity and social attraction in both studies . with regard to the results of step 2 , the adjusted rs had been increased largely . instead , the perceived appropriateness and entertainment value of target posts significantly predicted propinquity and social attraction . these results indicated that perceived entertainment value , in addition to perceived appropriateness , played an important mediating role in predicting closeness and social attraction . across two studies , participants perceived high - narrative disclosure messages as more entertaining , and the entertainment value predicted closeness ( propinquity ) and social attraction . for the effect of manipulated narrativity on propinquity , the 95% bootstrap confidence interval for the indirect effect of entertainment value , based on 5000 bootstrap samples , was [ 0.170.75 ] in study 1 , and [ 0.230.51 ] in study 2 . similarly , for effect of manipulated narrativity on social attraction , the 95% confidence interval for indirect effect was [ 0.170.64 ] in study 1 , and [ 0.220.50 ] in study 2 ( table 8) . the current studies utilized a unique design of stimuli that resembles a real - life browsing experience . it aimed to examine whether browsing social media can create a feeling of familiarity and ( even ) closeness toward strangers who publicly disclose self - information on social media . the results of study 1 and 2 consistently indicated that it is possible to develop a level of familiarity with a frequent discloser after browsing . perceived closeness to the target discloser remained relatively low , but the perceived closeness was higher compared to other disclosers who posted less self - information . more importantly , we aimed to examine the effects of disclosure intimacy and narrativity on perceived closeness and social attraction ( when the disclosure was likely to be perceived as appropriate ) . the manipulation of disclosure intimacy and narrativity worked largely in the direction of our expectation : it was perfect in study 1 ; but , in study 2 , even though we used the same stimuli , the manipulation of disclosure narrativity also slightly changed the level of perceived intimacy , and the effect size of manipulated intimacy on perceived intimacy was not as large as that in study 1 . when it comes to the role of disclosure intimacy , different from previous research ( baruh & cemalclar , 2015 ) , no negative relationships between disclosure intimacy and perceived closeness were observed in both studies . in study 1 , there was a positive effect of manipulated disclosure intimacy on perceived closeness ( the one - item measure ) , but a negative effect on social attraction . when we dropped the cases in which participants perceived the disclosure messages as inappropriate in study 1 , the positive effect of manipulated intimacy on perceived closeness turned stronger , and its negative effect on social attraction disappeared . however , no main effects of disclosure intimacy on closeness or social attraction were found in study 2 . this result indicated that , when disclosure messages are mostly appropriate , online participants are less likely , than lab participants , to be influenced by the level of disclosure intimacy . when it comes to the role of narrativity , we found a conditional positive effect on closeness in study 1 , and a significant positive effect on the social attraction in study 2 . this indicates that participants , when doing the study online , pay more attention to the overall feeling of coherence and narrativity than lab participants . in post - hoc analysis , we also found a mediating effect of perceived entertainment value : narrativity increased the entertainment value , and entertainment value turned out as an important predictor of closeness and social attraction . this is mainly due to the experiment settings : study 1 was conducted in lab , but study 2 was conducted online . lab participants tend to read the same stimuli more carefully than online participants , hence , in study 2 , the error rate of the familiarity with distractors was higher than that in study 1 and the manipulation check result of study 2 was not ideal . even with this drawback , it is practically meaningful to run the same study in online setting , as the online setting is closer to real - life sns browsing experience than the lab setting . much research has been done on the positive effects of public self - disclosure from a discloser s perspective , such as relieving loneliness ( buechel and berger , 2012 , groe deters and mehl , 2013 , lee et al . , 2013 ) , strengthening relational closeness ( burke & kraut , 2014 ) , increasing feeling of connectedness ( grieve et al . , 2013 , park et al . , 2011 ) , and improving subjective well - being ( lee , lee , & kwon , 2011 ) . less attention has been paid to the effects of public self - disclosure from a receiver s perspective ( edwards et al . , 2015 , 2015 ) , especially regarding the impression formation of the discloser in the zero - acquaintance or get - acquaintance paradigm . our research contributed to this research gap and provided a better understanding of public self - disclosure on social media and its effect on interpersonal relationships . the results of two studies contribute to the field of relationship formation on social media . prior research has mainly focused on the role of intimacy ; by examining the role of frequency , intimacy , and narrativity , we offer a more comprehensive understanding of the underlying processes . first , we showed that frequent posting on social media increases the feeling of familiarity and closeness in the zero - acquaintance paradigm . this could also explain the phenomenon of ambient awareness ( levordashka and utz , 2016 , thompson , 2008 , pp . 19 ) : merely by reading several posts that are embedded in a stream of other news , social media users can form an impression of the discloser and develop a feeling of closeness ( at least when the posts are perceived as appropriate ) . second , we examined the role of narrativity that has never been studied in this context . we found a direct or indirect positive effect ( via perceived entertainment value ) of narrativity on closeness and social attraction . it supported previous research claiming that entertaining posts on social media can bring people closer to each other ( lin et al . , 2016 , utz , 2015 ) , and addressed the role of humor in interpersonal relationship formation ( treger , sprecher , & erber , 2013 ) . the results on the effects of disclosure frequency and intimacy also contributed to the debate of familiarity - attraction link . for private conversations , a higher amount of self - disclosure often lead to more closeness ( a. aron et al . , 1997 ) , and created familiarity - based liking ( berger and calabrese , 1975 , zajonc , 1968 ) . recently , this basic principle has been called into question : norton et al ( norton et al . , 2007 ) proposed the less is more hypothesis that , especially in initial stage of interaction , more information about a person may lead to a higher degree of perceived dissimilarity , and therefore leads to less liking . our results showed that , for a feeling of closeness , a higher amount of self - disclosure is beneficial ; this supports the classic positive effect of self - disclosure on closeness . nonetheless , intimate self - disclosure , when perceived as inappropriate , seems to be detrimental for perceived social attraction . therefore , we recommend social media users to be cautious when disclosing intimate self - information publicly on social media , but it would be nice if one can disclose self - information in a narrative way . practically speaking , frequent posting on social media is good for others to have a feeling of familiarity with oneself , but it does not necessarily lead to social attraction . especially when such public posts are perceived as inappropriate or lead to negative beliefs , posting may decrease social attraction . first , we purposefully used the design that five posts of the target discloser were embedded in a news stream of fifteen posts . we are not sure how a different number of posts in the stimuli may alter the results . for example , posts in the stimuli can pop up one after another in the experiment , to resemble the process of news updating in one s news stream . second , there were some problems with the manipulation : the result of manipulation check in study 2 was not ideal , and we doubted if the manipulation was strong enough . the manipulation checks were measured with the target posts provided on the same page , but closeness and social attraction were not . when participants paid less attention to the stimuli before manipulation check , they were less likely to be influenced by the manipulation when rating closeness and social attraction . it was not easy to disentangle and manipulate these variables in experiments , though they are distinct concepts in theory . third , there were a few problems with regard to the measures of perceived intimacy and one - item closeness . in addition , even though one - item measure of closeness was popular ( arthur aron et al . , it was more convenient for participants to rate it multi - times for examining h1 , and we purposefully added another multi - item measure of propinquity for further examination . in summary , we examined the role of public self - disclosure on interpersonal impression formation by conducting one lab study and another online replication study . both studies indicated that a higher frequency of self - disclosure on social media is beneficial for others to create a feeling of familiarity with oneself . however , the effects of disclosure intimacy and narrativity on the feeling of closeness and social attraction are somehow influenced by other factors such as perceived appropriateness and entertainment value . on social media , it is usually good ( at least does no harm ) if one can disclose self - information in a narrative way , as it increases entertainment value ; but one has to be cautious when disclosing intimate self - information that might be perceived as inappropriate by others , which can decrease one s perceived social attraction .
on social media , users can easily share their feelings , thoughts , and experiences with the public , including people who they have no previous interaction with . such information , though often embedded in a stream of others news , may influence recipients perception toward the discloser . we used a special design that enables a quasi - experience of sns browsing , and examined if browsing other s posts in a news stream can create a feeling of familiarity and ( even ) closeness toward the discloser . in addition , disclosure messages can vary in the degree of intimacy ( from superficial to intimate ) and narrativity ( from a random blather to a story - like narrative ) . the roles of disclosure intimacy and narrativity on perceived closeness and social attraction were examined by a 2 2 experimental design . by conducting one lab study and another online replication , we consistently found that disclosure frequency , when perceived as appropriate , predicted familiarity and closeness . the effects of disclosure intimacy and narrativity were not stable . further exploratory analyses showed that the roles of disclosure intimacy on closeness and social attraction were constrained by the perceived appropriateness , and the effects of narrativity on closeness and social attraction were mediated by perceived entertainment value .
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Proceed to summarize the following text: the scientific - technological development that has occurred in the last half of the twentieth century determined important changes in the course of the disease , being the pathologies of chronic character the leading cause of death in modern societies . however , this increase in longevity brought another kind of concerns related to comorbidities and limitations that arise as a result of living with a chronic illness . in general , we live longer but this increase in life expectancy does not always correspond to better quality of life . in this context , issues related to quality of life have been gaining special importance and are widely studied in various contexts , particularly in oncology . cancer which is one of the leading causes of death worldwide is one of the most dreaded diseases in the present [ 2 , 3 ] . the strong physical , psychological , social , emotional , and spiritual impact reminds the person of one 's vulnerability and leads one to wonder about the meaning of life , and that may cause suffering . recent studies have shown that the concept of quality of life is closely associated with self - perception about wellbeing , with the satisfaction with life and happiness . in this context , health professionals , particularly in the context of palliative care whose mission is to promote the best possible quality of life for the patient and family have been encouraged to investigate and find strategies that allow the person to find moments of happiness and wellbeing , even in difficult circumstances , as is the experience of a cancer or terminal disease . happiness is an essential component of quality of life , to the point that the world health organization recognized it as part of the concept of health . however , as documented by other authors , little is known about its significance for people living with advanced cancer . after a comprehensive review , we found some studies where the concept was explored in patients with cancer [ 7 , 9 ] , parkinson 's disease , and alzheimer 's disease . yet none of these was held in portugal or in the context of palliative care , where the theme is especially important . these studies are recent and emerged in the last 10 years which suggests a growing interest in the topic in the context of health . previous research suggests that happiness is a complex multidimensional concept , influenced by individual and cultural factors , with strong repercussions in all dimensions of human life . in a recent study , the authors concluded that happiness is perceived by patients with advanced cancer as a fundamental precursor to quality of life , helping them to live the remaining time in the best possible way . the health literacy is defined as a person 's ability to obtain , process , and understand information on health and how this process can help one in decision - making [ 12 , 13 ] . the research has suggested that health literacy helps people to make informed and aware decisions , which are extremely important factors in an increasingly complex health care system . in addition to these considerations , efforts have been alerting health professionals to the fact of low literacy , particularly in cancer patients , being associated with lower levels of welfare , higher levels of anxiety and depression , erroneous conceptions about the disease , and , consequently , lower adherence to the therapeutic regimen and less satisfaction with the care provided [ 12 , 1416 ] . the information given to the patient as well as the decisions to be taken , particularly in contexts of severe disease , is difficult and with repercussions , not only for professionals but also for the patient and family . in this context , nurses assume a privileged position , because the proximity to the patient allows them to be more attentive to their needs and problems . assuming that happiness and health literacy are important to patients with advanced cancer , we conducted the present study , which aims at analyzing ( a ) the perception portuguese cancer patients undergoing chemotherapy have about their health status , ( b ) the satisfaction they have with the information relating to their health status , ( c ) their level of happiness , and ( d ) how these patients perceive their own future . an observational , cross - sectional , exploratory , and descriptive study was developed , aiming at understanding how information about the health status and their belief systems influence the vision of the future and happiness of people undergoing chemotherapy . portuguese cancer patients undergoing chemotherapy were the chosen population for this study . due to the impossibility of accessing all these patients , it was decided to restrict the study to patients who undergone chemotherapy in the day hospital ( ambulatory care ) of a central hospital in northern portugal . the reason of this choice is due to the fact that this is a large department , with patients from different age groups and also with different malignancies , facts that we considered important in order to obtain a representative sample . the following inclusion criteria were defined : ( a ) 18-year olds , ( b ) portuguese nationality , ( c ) malignant disease diagnosed , and ( d ) undergoing chemotherapy . for ethical and legal reasons , all patients under 18 and with cognitive limitations , as well as those undergoing chemotherapy for the first time were excluded from the study . data were collected between may 16 , 2012 , and july 16 , 2012 , according to instructions and availability of the service where the study was conducted . in order to assure the respect for ethical principles , particularly the nonmaleficence , and so as not to interfere with the care and service dynamics , data were collected during the treatments , which generally lasted several hours . in this period all patients undergoing chemotherapy were approached ( regardless of the purpose of this be curative or palliative ) , up to the limit of the sample under study , and according to the inclusion and exclusion criteria defined . the data collection instrument was built by a self - completion questionnaire , consisting of two distinct parts . in the first part , information to sociodemographic , clinical , and faith was requested . from the data collected here , we analyzed the following variables : gender , age , marital status , education , household composition , diagnosis , time on chemotherapy , purpose of chemotherapy , religion , religious practice ( practicing or not practicing ) , and opinion about god . in the second part of the instrument , which resulted from an adaptation of the permanent survey on social attitudes of the portuguese , five closed - answer questions ( likert scale ) related to the perception of health status , satisfaction with the information provided on the state of health , vision of the future , fear of the future , and the level of happiness ( dependent and ordinal variables ) the final instrument was reviewed by an expert panel constituted by the authors and three nurses ( an expert in the validation of scales , a spirituality researcher and a palliative care expert ) . this pre - test concluded that the instrument was simple , clear and objective , which is why it was not necessary to make changes to it . the research project was previously approved by the ethics committee and the administration council of that institution . the information was analyzed using the statistical package for the social sciences program , version 18 for windows , and descriptive statistics were used . the participants in the study ( n = 92 ) were approximately 54 years old ( mean = 54.17 years ) . the youngest patient was 18 and the oldest 79 years . in the total of respondents , 51.10% were male and 48.90% female . most of them lived only with spouse ( 46.70% ) and 38.00% with the spouse and children . despite being with a malignant disease in an advanced stage , 2 patients ( 2.20% ) reported living alone . regarding the qualifications , we found that the majority of subjects had only 4 years of education ( 47.90% ) , which corresponds to the elementary education in portugal . from a clinical standpoint , it was found that most respondents had bowel cancer ( 25.29% ) and breast cancer ( 20.70% ) . of the total respondents , 54.30% were in chemotherapy with curative intent and 45.70% in chemotherapy with palliative goals . the majority of individuals in palliative chemotherapy were males ( 29.30% ) and had bowel cancer ( 10.90% ) . patients in curative chemotherapy were essentially females ( 32.60% ) and had breast cancer ( 17.40% ) . in the item about religious beliefs , patients were questioned about their religion , religious practice , and beliefs about god . six patients did not respond to the questions religion and religious practice . the majority of claims to be catholic ( 93.00% ) and it was found that there is no great disparity between those who admit having a regular religious practice ( 51.10% ) compared to those who do not have it ( 48.80% ) . however , those who have a regular religious practice are those with more certainty ( 40.50% ) . only one patient reported not believing in god . when asked how they regarded their health status at that time , most patients ( 51.10% ) rated it as neither good nor bad . despite this neutral position , 30.40% considered that their health was good , being relatively low the percentage of patients who consider their health as bad or very bad ( 13.30% ) ( table 1 ) . with regard to how they saw their future , few have considered it very good ( 7.60% ) , slightly higher than that found for the group of patients who considered their health status as very good ( 4.30% ) . in general , despite the difficulties imposed by the disease , the vision of the future is always more optimistic than the perception of the current state of health : 35.90% perceives the future as good and although 3.30% consider their health very bad , only 2.20% believe that the future will be itself very bad . however , the great majority is afraid of the future ( 63.00% ) and 18.50% reported being very frightened . with regard to the degree of satisfaction with information on the health status and despite not having analyzed the information they had it was found that the great majority of the patients felt very satisfied ( 44.60% ) or satisfied ( 29.30% ) with the information about their health status . in the last issue , participants were encouraged to think about all aspects of their life and assess their current state of happiness . in spite of the majority considering to be fairly happy ( 57.60% ) , it is important to note thatthere is a significant group of patients who considered themselves to be very happy ( 23.90%);very few considered themselves unhappy ( 2.20%);among those fairly happy considered , 64.30% were in palliative chemotherapy;16.70% of the patients in palliative chemotherapy considered themselves as very happy people . there is a significant group of patients who considered themselves to be very happy ( 23.90% ) ; very few considered themselves unhappy ( 2.20% ) ; among those fairly happy considered , 64.30% were in palliative chemotherapy ; 16.70% of the patients in palliative chemotherapy considered themselves as very happy people . analyzing these items , it was found that there is a large percentage who considers their health as good or very good ( 35.20% ) , which is quite interesting considering they are individuals with a very serious disease . however , it is important to note that those who perceive their health status as bad or very bad are mostly in palliative chemotherapy ( 14.60% ) . with regard to the vision of the future and happiness , it was found that satisfaction with information about the health status exerts an important influence . in general , patients who considered themselves well informed about their health status reported having no fear of the future ( 24.20% ) , and , despite living with a serious life - threatening cancer disease , they saw it as good ( 22.00% ) . on the other hand , when we perform the summation of the responses of those who consider themselves less informed about their health status , we found that 17.60% are afraid of the future . still regarding the variable information , it was found that those who consider themselves more informed about their health status , are also considered happier . in fact , 46.70% feel satisfied or very satisfied with the information relating to their health , considering themselves fairly happy or very happy . those more informed about their health status , are also those who fear the future less ( 24.20% ) . finally , when the variables of the future vision and beliefs about god were crossed ( table 3 ) , it was found that those who believe in god and have no doubts about his existence see the future as neither good nor bad ( 25.30% ) or good ( 25.30% ) . similarly , these are also the individuals who mostly refer not being afraid of the future ( 23.10% ) or only slightly ( 19.80% ) . the results show us the perspective of a group of patients with advanced cancer in chemotherapy . previous studies have shown that cancer is one of the most feared diseases by modern societies , with strong impact on the quality of life of the human person , particularly during chemotherapy treatment . other studies have been documented that quality of life is closely linked to the perception that the person has in relation to one 's wellbeing , life satisfaction , and happiness . the study confirms this vision but it also adds new data , suggesting that religious beliefs and health literacy may have an important contribution in promoting wellbeing and patient 's quality of life . although all participants have an advanced cancer , some of them in palliative chemotherapy , the vast majority has an optimistic view of the future and considers themselves as happy persons . the majority reported being afraid of the future but , despite this , their vision of the future has always been more positive than their perception about health status . this optimistic vision is associated with hope , a concept closely associated with wellbeing and happiness and essential in the care of people with cancer . previous studies have shown that the need to maintain a positive thinking is crucial because it helps the person to deal with the suffering caused by the disease , to restore the meaning of life and find purpose to continue [ 1921 ] . it was also found that religious beliefs can have a significant impact on how a person views the future . although the majority of respondents are catholic , as fits the profile of portuguese citizens , beliefs about god have varied . the study revealed that those who believed in a higher entity and not had any doubts about his existence had less fear of the future . these data confirm those obtained by other authors and show that religious and spiritual beliefs are closely associated with the concept of quality of life , helping the person to maintain hope , and dealing with the uncertainty of the future in a more effective way [ 2022 ] . in addition to this positive view tendency , most respondents considered themselves happy or very happy , noting that a significant group of these patients was in palliative chemotherapy . this seems to us to be an important indicator of the quality of life of this group of patients and therefore alerts us to the importance of providing care in a holistic perspective , in order to provide patients with moments of wellbeing and comfort even at the time of disease . happiness is a complex and multidimensional experience and , in this sense , health professionals should strive to meet the needs of the human person in all dimensions of life : physical , psychological , social , emotional , and spiritual . in addition to these data , this study has also shown that the information that the patient has about their health status and the way how one processes and perceives that information may have an important influence on how the future is looked and consequently on their happiness . we found that patients more satisfied with the information regarding their health status had less fear of the future , regard it as better , and consider themselves happier . these data are corroborated by a study carried out in 2003 with cancer patients , where the author concluded that patients who knew the effects of treatment had a more positive perception of their health status and higher levels of hope . in the present study , we did not assess which information was given to participants , but we found that the information given was enough to answer their questions and doubts , helping them to live that period of their lives in the best possible way . in this context , we can say that more important than what information to give the patient is how and when to give this information . the low health literacy which includes not only the information given , as well as how the person understood and perceived this information is associated with poorer health outcomes , including lower adherence to the therapeutic regimen , misconceptions about the disease , and higher levels of depression and anxiety [ 12 , 1416 ] . the study shows , however , some limitations . the sample type ( not randomized ) does not allow the inference of the results for the population moreover , the nationality of the participants in the study may have influenced the results . generally , the portuguese are seen as optimistic people , who tend to face the future with hope , as suggested in previous studies [ 20 , 21 , 25 ] . for practical reasons , the study was limited to patients who carry out chemotherapy in day hospital . within this group , although ethical motivations should prevail , it is important to note that these criteria limit the results . thus , the data refer to the sample under study and future generalizations should be made with caution . despite these limitations , the present work encourages health care professionals to find ways of helping cancer patients live the period of the disease in the best possible way . the information transmitted and how the patient processes this information , along with the religious beliefs , can act as important coping mechanisms , helping the person to deal with suffering , to look to the future with hope and be happier . although all participants in the study had severe malignant disease , some of which in palliative care , it was found that in general they are happy people with an optimistic view of the future . information on health status , commonly referred to as health literacy , plays an important role in these results , showing that patients who feel well informed , fear less the future , perceive it as better and consider themselves happier . despite the study 's limitations , particularly with regard to the type of sampling ( which is not random prevented us infer the results to the population ) , we believe that the results are important . the study supports scientific evidence , particularly the idea that health literacy and promotion of happiness can have a strong impact on people 's quality of life with advanced chronic disease , helping them to live the moments of crisis in the best possible way . the study has thus important implications for future research and for clinical practice , showing the importance of providing care in a holistic manner , directed to the ill person and not just to fight the disease . we live in a time characterized by technology and that mechanized vision should never be extended to the nursing care which is holistic and human in its nature nurses must therefore be alert and seek to collect a range of information that may be important coping mechanisms such as religious beliefs , family support , and information about the health status . another important aspect passes through the demystification of some wrong information people have about the disease and about cancer treatments , and which may affect how a person lives in the present and sees the future . results show that information about the state of health is not only a fundamental human right , but also an important promoter of happiness , wellbeing , and therefore quality of life . thus , it is absolutely essential that nurses help fight the fear associated with the disease and treatments , trying to be available to listen , answer questions , provide supporting structures , or refer to other professionals when needed . however , simple gestures such as praying with the ill person , the therapeutic touch , listening , or teaching the family can also be extremely important in helping to reduce the patient 's suffering and promoting their happiness and wellbeing . happiness , in turn , is determined by a set of circumstances where the health literacy and religious beliefs can play a key role . both concepts health literacy and happiness are closely associated with hope . however , more studies are needed to relate the association between these concepts and analyze the indexes of happiness based on the type of information given .
cancer is a dreaded disease that affects all dimensions of human life . in this context , issues related to the quality of life as happiness , perception about health status , or health literacy are important . this study aims to analyze the following topics the perception : the portuguese cancer patients have about their health status while undergoing chemotherapy , the satisfaction with the information relating to their health , their level of happiness , and their vision of the future . an observational , cross - sectional , and descriptive study was developed . data were collected between may and july 2012 in the day hospital of a central hospital in northern portugal . the sample was composed of 92 cancer patients who were asked to answer a questionnaire during chemotherapy . the results indicate that , despite this life - threatening disease , patients consider themselves fairly happy and have an optimistic view of the future . information about their health condition and religious beliefs was important coping mechanisms to help dealing with the suffering caused by the disease . the study highlights the importance of providing care in a holistic way . nurses must be alert and available to listen , answer questions , provide supporting structures , or refer to other professionals when needed .
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Proceed to summarize the following text: pancreaticobiliary cancer remains a lethal disease where treatment continues to be a major oncologic challenge . most patients present with advanced disease in which survival is dismal . for patients diagnosed early with small tumors , surgery offers a chance of cure . in an attempt to diagnose these tumors at an early stage , many studies have evaluated the use of tumor markers , but so far , due to low sensitivity and specificity , the results have not been promising . the high metastatic potential of pancreatic cancer has led researchers to investigate its ability to invade the basement membrane and extracellular matrix ( ecm ) allowing it access to capillaries and lymphatics . one process that has been shown to be involved in invasion of most types of cancer is the urokinase - type plasminogen activator system . upa is a serine proteinase that is best known for catalyzing the conversion of inactive plasminogen to the active proteinase plasmin . upa and its receptor upar have been demonstrated to be involved in tumor invasion , growth , and metastasis . independently or through the activation of plasmin , upa can degrade ecm , activate matrix metalloproteinases , mediate the release of growth factors ( including transforming growth factor , fibroblast growth factor , vascular endothelial growth factor , insulin growth factor , tumor necrosis factor , and hepatocyte growth factor ) , stimulate cellular migration , induce chemotaxis , and promote angiogenesis [ 3 , 4 ] . since the 1980s when upa was postulated to have a role in tumor invasion , there have been multiple studies evaluating it as a prognostic marker of cancer . it was first studied in breast cancer where it has been shown as an independent prognostic marker for predicting survival second only to lymph node status . following the initial results in breast cancer , upa overexpression was shown to confer a worse prognosis in many other cancers including colorectal , esophageal , gastric , hepatocellular , prostate , sarcoma , and head and neck squamous cells among others [ 612 ] . the importance of the upa activator system has also been demonstrated in pancreatic cancer [ 1318 ] . the first study to show overexpression of upa in pancreatic cancer was by takeuchi et al . in 1993 . he demonstrated by immunohistochemical staining that 78% of pancreatic cancers overexpressed upa and this overexpression correlated with decreased survival . another study by cantero et al . in 1997 showed that concomitant overexpression of upa and its receptor upar correlated with shorter survival times . we detected upa overexpression in pancreatic intraepithelial neoplasia ( panin ) and histologically normal ducts / acini that are in the immediate vicinity of the tumor . upa was also found in the vessels of tumor stroma suggesting that upa is in circulation , and therefore should be detectable by serum analysis . it allows further evaluation through imaging of the ductal system and allows to sample pancreatic juice and brush biopsy cytology for further analysis . ercp has also been studied in small numbers to screen high - risk patients with a family history of pancreatic cancer , and it demonstrated that cytologic brushings showing dysplasia correlate with the pathologic specimen after resection [ 20 , 21 ] . despite combination of sampling with brush cytology , fine needle aspiration , and biopsy , the sensitivity of all three combined is only 62% . most practitioners perform only brush cytology which by itself has a sensitivity of only 30% in the diagnosis of pancreaticobiliary malignancies . we hypothesized that molecular markers such as upa may improve our ability to differentiate malignant from benign pancreaticobiliary strictures and assist us in planning therapeutic strategies . we performed a feasibility study of patients undergoing ercp for confirmed or suspected pancreaticobiliary cancers to determine whether upa overexpression could be identified from cytologic brushings . serum levels of upa were measured in this patient group and compared to healthy subjects . eleven patients with known or suspected pancreaticobiliary malignant strictures secondary to either pancreatic cancer or cholangiocarcinoma as determined clinically by radiologic studies were consented to have cytologic brushings obtained during ercp for evaluation of expression of upa . at ercp , one slide was routinely stained for h&e to identify the presence of epithelial cells and the other was stained for upa in a blinded fashion . presence and type of cancer were determined by biopsy and reviewed by a pathologist . reagents used in the immunohistochemical procedure including blocking solution , secondary antibody , and vectastain elite abc reagent were purchased as the r.t.u vectastain universal elite abc kit ( vector laboratories , burlingame , ca ) . color - development reagents were purchased as part of a dab substrate kit for peroxidase ( vector laboratories , burlingame , ca ) . the dab substrate kit contained the reagents required to make a working solution of 3,3-diaminobenzidine ( dab ) for staining tissue sections . upa expression in cytologic brushings obtained during ercp was evaluated by immunohistochemistry ( ihc ) . the upa1 primary antibody was used at a concentration of 20 g / ml . cells were blocked with normal horse serum ( 2.5% ) for one hour and incubated overnight at 4 celsius with the primary antibody . after gently rinsing the cells with triton - tbs , they were treated with the secondary antibody ( r.t.u . biotinylated universal antibody and anti - rabbit / anti - mouse igg made in horse ) , gently washed and treated with vectastain elite abc reagent . a slide of cytologic brushings was also stained by the same procedure , except that an irrelevant isotype igg was substituted for the primary antibody as a negative control . staining intensity for upa was classified as 03 + ( 0 absent ; 1 + , weak ; 2 + , moderate ; 3 + , strong ) . serum levels of upa were analyzed using an enzyme - linked immunosorbent assay ( elisa ) . correlation between ihc grade and serum levels of upa was estimated with the spearman rank correlation coefficient . all tests were carried forth using sas 9.2 , sas institute inc . , cary , nc . given n = 11 subjects per group and assuming exchangeability under the null hypothesis , the wilcoxon rank - sum test would be able to detect approximate shift alternatives of roughly 0.6 ng / ml , 1.3 ng / ml , and 1.7 ng / ml given 0.80 power and common standard deviations under the null hypothesis of no difference of 0.5 ng / ml , 1.0 ng / ml , and 1.9 ng / ml , respectively . 8 patients had pancreatic adenocarcinoma and 3 had cholangiocarcinoma . when immunohistochemistry for upa was performed on the cytology specimens , six of the eight patients ( 75% ) with pancreatic cancer ( figure 1 ) and two of the 3 patients ( 67% ) with cholangiocarcinoma overexpressed upa ( figure 2 ) . the two patients with pancreatic cancer who did not overexpress upa were being treated with chemotherapy and underwent ercp for biliary stent exchange . the mean serum upa in the 11 patients with cancer was 1.27 ng / ml ( sd = 1.54 ) . the mean serum upa in the 11 normal healthy individuals was 0.56 ng / ml ( sd = 0.16 ) . serum analysis demonstrated a 2-fold higher concentration of upa in the pancreaticobiliary cancer patients compared to the healthy controls ( p = .0182 ) . table 1 compares the level of upa staining in cytologic specimens to serum levels of upa for all 11 patients . the ihc grade for upa staining correlated with serum levels for upa ( r = 0.72 ; p < .0001 ) . a definitive diagnosis of cancer can be difficult in patients with pancreaticobiliary strictures with no obvious mass on imaging studies . we have previously shown that upa is an early event in the malignant transformation of pancreatic cancer . therefore we sought to determine the feasibility of determining upa expression in cytologic brushings of established malignant strictures to evaluate its potential as an adjunct to ercp in diagnosis of pancreaticobiliary malignancies . in our small sample , we found it is possible to identify overexpression of upa in cells obtained from brushings during ercp . upa overexpression ( 72.7% ) appears to be a marker for pancreatic or biliary cancers . although there is no current literature to show that chemotherapy directly inhibits upa synthesis , we have postulated that this treatment impedes growth , and thus decreases upa expression . the relationship between upa overexpression and cholangiocarcinoma has not been studied , but in two of the three ( 66% ) samples , the immunohistochemical analysis of the cytologic brushings showed overexpression . as ercp is an invasive technique with potential morbidity , we also looked at serum upa in our patient population , and it was elevated in patients with pancreaticobiliary cancer . the serum levels correlated with the ihc grade of upa in tissue ( p < .001 ) . serum upa levels have been evaluated in breast cancer by rha et al . , who showed blood upa levels correlated with those of tissue . because our study contains only a small number of patients , the value of serum upa as a tumor marker needs to be further evaluated in large numbers of patients including patients with chronic pancreatitis before it can be compared to the standard tumor markers such as ca19 - 9 . currently there are no established screening tests for pancreatic cancer . the united states preventive services task force ( uspstf ) has recommended against routine screening for pancreatic cancer based upon the lack of data demonstrating its clinical value in the general population . familial pancreatic cancer ( fpc ) accounts for up to 10% of patients with pancreatic cancer . iii , and patients with panin iii have been considered for pancreatectomy by some groups . surveillance by ercp and endoscopic ultrasound is performed in an attempt to identify patients with fpc who have developed panin , but the diagnosis is difficult . evaluating upa overexpression from ercp obtained cytologic brushings may have potential application in screening high - risk patients by identifying either panin or invasive pancreatic cancer . although ercp arguably remains the golden standard for pancreaticobiliary evaluation , there is a risk of complications associated with ercp including pancreatitis ( 5.4% ) . endoscopic ultrasound ( eus ) with eus - guided fna has emerged as an extremely sensitive and specific method of diagnosing pancreatic cancer and can detect small lesions more effectively than conventional imaging . for the diagnosis of pancreatic cancer , as we have shown that upa staining can be performed on brush cytology , it can be done on fna specimens as well and may improve diagnostic accuracy . as targeted therapy has stepped to the forefront of cancer research , molecular markers have moved from their traditional roles of diagnosis and staged to possible aims of treatment . already upa has been evaluated as a potential target for treatment to decrease the invasive and metastatic activity of pancreatic tumor cells . evaluated an anti - upar monoclonal antibody in mice and found that it significantly decreased tumor growth , hepatic metastases , and retroperitoneal invasion . small molecule inhibitors of upa have been evaluated in a fibrosarcoma model in mice resulting in decreased metastases and prolonged survival . these results suggest that detecting overexpression of upa in pancreatic cancers may not only be prognostic and diagnostic but may also direct treatment in the future . a serious limitation of the current study is its small sample size and lack of an adequate control group precluding meaningful analysis . in this study , a control group of healthy subjects for the analysis of upa in the serum was obtained . there are obvious ethical issues in obtaining brushing from healthy volunteers and it would appear more suitable to utilize patients with benign pancreatic and biliary strictures . we elected to exclude potential control groups such as those presenting with obstructive jaundice from benign pancreaticobiliary strictures . the possibility that some of these patients harbored occult malignancy would be difficult to discern without long term follow up . even in patients with choledocholithiasis , there may be a potential for the associated inflammatory process to elevate upa levels . thus , we performed this feasibility study in patients with known or suspected pancreaticobiliary malignancies by brush cytology to corroborate our prior findings in resected pancreatic cancer specimens . we performed a feasibility study to determine whether upa overexpression could be identified from cytologic brushings of patients undergoing ercp for confirmed or suspected pancreaticobiliary cancers . serum levels of upa were measured in this patient group and correlated with upa overexpression . larger studies involving all forms of pancreaticobiliary pathology need to be performed before upa overexpression can be used either as a serum or a cytological tumor marker , especially those with no identifiable masses . also comparison with other tumor markers , that is , ca 19 - 9 and cea will be required to substantiate the role of upa as a diagnostic or prognostic tumor marker .
we have previously demonstrated that upa is overexpressed in pancreatic tumors . in an attempt to diagnose these tumors earlier , we sought to determine whether upa could be identified in endoscopic retrograde cholangiopancreatography obtained brushings in patients with malignant pancreatic and biliary strictures . secondarily , upa was measured in the serum of this patient population . upa overexpression was identified in the cytologic tissue in 8 of 11 patients ( 72.7% ) . serum analysis demonstrated a 2-fold higher concentration of upa in the pancreaticobiliary cancer patients ( 1.27 versus 0.56 ng / ml ; p = .0182 ) . also , upa overexpression correlated with serum levels ( p < .0001 ) . this study confirms that upa can be detected in the ercp cytologically obtained tissue and is frequently present in a higher concentration in the serum of pancreaticobiliary cancer patients . a larger sample size will be required to address its value as a sensitive marker for the diagnosis of pancreatic or biliary cancers .
You are an expert at summarizing long articles. Proceed to summarize the following text: in the majority of industrialized countries , patient identification lies at the heart of many of the concerns relating to electronic processing of health information . in august 2004 , the french government decided by law to start a national project for an electronic health record called the it is designed to promote health care coordination , enhance communication of health information , and reduce iatrogenic accidents . the information system corresponding to this project is still under construction . regarding the patient identifier , one of the proposals has been to use the existing social security number ( ssn ) . however , civil society and bodies representing citizens ( patients ' associations , those defending individual liberties , national councils of medical associations ) are worried , quite rightly , about the security of medical information should the ssn be used as an identifier in the field of health . they are afraid that medical information may be linked with other information ( social , economic , financial , employment ) often identified by the ssn . this is one of the reasons why the government decided to postpone this project ( november 2007 ) . nevertheless , in december 2007 , a working group was mandated by the government to propose , as soon as possible , a solution for the hin , which will be used not only for the dmp project but also for health in general . through this paper , we first propose a method which aims both to reassure citizens ' representatives regarding the security of medical information and to give the opportunity to the french government to utilize a derived ssn with the same reliability as the ssn . a solution based on the utilization of anonymity techniques secondly , we analyse the conditions necessary to make this first solution interoperable at the european level . in an increasing number of countries , patients have direct access to their medical records ( mr ) , and some countries including france have decided to provide a personal medical record ( pmr ) to the patient . we define mr as patients ' medical information recorded by the medical practitioner under his or her own responsibility and ideally electronically signed by him / her in order to authenticate the provider ( health professional ) and to prevent any modification of its content . in contrast , the pmr is personally supervised by each patient , who has the right to mask any information he / she does not want to be read . as the owner of this pmr , the patient determines who can gain access to his / her record . of course , patients are very worried that information concerning their private lives may be disclosed . such concerns are increasing as more sensitive medical details , such as psychiatric records , hiv status , and genetic information , are stored in their pmrs . in france , to enhance the coordination of care , the pmr 's data will be stored in national shelters . so patients need to be sure that medical information may not be linked with other information ( social , economic , financial , employment ) also identified by the ssn , if the ssn was used as an identifier for the pmr . many other countries have chosen a homogenous national patient identification system ( e.g. , northern european countries such as denmark , finland , luxembourg , the netherlands , belgium , the united kingdom , and ireland ) , and the former countries in this list are even using the same identification number for other fields than health care . apart from belgium , the above - mentioned countries implemented a unique patient identifier some time ago , and the citizens are used to it and to the creation of national health databases . however , in the united kingdom , patients are reassured because there is no national shelterer and medical records are stored by the general practitioner who can not transmit or receive any information regarding a patient without his / her consent . in southern european countries identification is often organized at a regional level ( spain and italy ) and patients are more familiar with the creation of large health databases organized at a federal level as in other countries ( canada and the usa ) . to reassure french patients regarding the security of their medical data which will be stored at a national level , we propose in this paper the creation of a secure patient identifier which should be different from the ssn to avoid linkage with other data , but which should be as reliable as the ssn . this proposed solution could be used by any country needs to implement a unique health identification number . as we said before , it is perfectly possible to preserve the confidentiality expected by the patient by putting in place anonymity procedures [ 6 , 7 ] such as those adopted by the institut de veille sanitaire ( the french institute for public health surveillance ) on the recommendation of the french national commission for data protection , in the context of followup procedures for the 30 diseases subject to mandatory reporting ( including aids ) . unlike encryption methods that must be reversible to allow the legitimate recipient to decode the message , unidirectional hashing techniques , such as the standard hash algorithm in its modified version sha-2 ( sha-2 is considered significantly stronger than sha-1 , although somewhat slower : nsa and nato recommend it in the suite - b package ( ecc , aes and sha-2 . ) ) , are irreversible . hashing produces a strictly anonymous code ( it is not possible to retrace the patient identity ) that is always the same for a given individual and thus ensures that patient data can be linked . there are many medical applications including the creation of national databases ( such as those relating to the national followup of infected persons approximately 100 000 patients which are an excellent example of how it can help epidemiological research , with complete patient approval ) as well as regional and interregional databases in many areas ( cancer , perinatal diseases , genetic diseases ) . this system , which is based on the hash coding of the social security number , the gender and the date of birth , has also made it possible to link all standardized hospital discharge abstracts , classified into french diagnosis - related groups at the french national level , and to link the data of the national medical insurance information system . an anonymity procedure based on hash coding is also used to chain patient files in switzerland . similar solutions , also derived from the irreversible encryption of the unique social security number , have been proposed in belgium and new zealand . in the case of the pmr , the situation is no more complex because similar requirements must be met . the french commission for data protection , and associations of patients and healthcare professionals demand the confidentiality of personal information contained in the pmr . public health bodies or individuals need to have access to these data , particularly when the patient has given express consent . the french commission for data protection , and associations of patients and healthcare professionals demand the confidentiality of personal information contained in the pmr . public health bodies or individuals need to have access to these data , particularly when the patient has given express consent . regarding confidentiality , insofar as the social security number ( ssn ) could not be reconstituted using the health identifier number ( hin ) , the link between them would be broken . another advantage of using hash coding is that it meets the criterion of being focused ( created and maintained solely as a support for health care ) . this is one of the main criteria of a universal health care identifier that were published by the american society for testing and materials , a standards development organization accredited by the american national standards institute , in the standard guide for properties of a universal healthcare identifier . this solution satisfies the demands of french commission for data protection in that the ssn would be rendered anonymous by a trustworthy third party and would thus permit the generation of a health identifier number ( hin ) known by the patient . this supposes that the hin would be included in the patient smart card , used for administrative and financial purposes ( reimbursement of medical care ) . for french patients associations , the interest and importance of printing the health identifier number ( hin ) on the patient smart card lie in the fact that it would mobilize and sensitize patients , and increase their awareness that the identifier is different from the ssn . in fact , if the ssn was rendered anonymous using a hash algorithm , there would be no link between the new number and the personal details of the individual , which is not the case for the ssn . nevertheless , it seems to us that this solution is an illusion in terms of security and reliability because the correspondence between the ssn and hin would be set out on the patient smart card and would therefore also be known by the health workforce : the ssn is needed in the smart card for reimbursement purposes . with regard to methodology , to provide anonymity safely requires double hashing , because anonymity is not guaranteed by a single hashing process [ 6 , 7 ] ; two hashing keys must be used to obtain complete anonymity . as a consequence , this solution must be completed by the following principles . according to figure 1 , the health professional can provide to the portal of the application a coding hin obtained through reversible encryption . the hin will itself be transformed into a stocking hin in the same way that the ssn was transformed into an hin , but with a different key ( see figure 1 ) . this portal can then transmit this code to the data - processing shelterer safely and reliably . if a person manages to get the ssn of a patient and uses the hash function , he has to know the key used by the trustworthy third party to obtain the hin of the patient . however , as the ssn and the hin of the patient will both be included in the smart card of the patient and used by health professionals , there is a risk of a breach of privacy in health structures . the aim of hash coding of the hin by the portal ( with a more different key than that used for hash coding the ssn ) is to obtain an anonymised identifier ( stocking hin ) before sending it to the data processing shelter . it also seems fundamental to us that prerequired conditions be imposed in terms of security and confidentiality regarding access to medical data in health structures . this raises several concerns , the first of which is related to the authentication of health - care professionals . the solution would be a smart card attributed to professionals in both the private sector and public hospitals . in france it is not only about controlling the right of access to different applications , but also about ensuring the security of local hard disk storage , preventing direct access to the databases , and guaranteeing the security of external archives ( band and cd - rom ) . lastly , we are also very concerned about the security of exchanges among health professionals . we must point out that many health - care professionals require access to medical records from home , for example , or when on call . a similar situation arises when records are forwarded to doctors during vacations through the internet . we also know that some data processing specialists or computer science experts need access to health structure data for application maintenance . we then strongly recommend the use of networks like virtual private network , in addition to the professional authentication card , as a solution to the problem of the security of data exchange . mobility must not compromise healthcare for european nationals . residents of one member state travelling to or working in another member state should have the same right to high - quality healthcare as all other europeans ( see , e.g. , regulation ( ec ) no 883/2004 ) . it is thus very important to ensure interoperability between the patients identifiers in the different european countries . however , in europe , each country has its own patient identifier with very different structures and contents , and which may not always be derived from an ssn . as a consequence , the use of the ssn alone ( or a derived ssn ) to generate the hin in european countries would not solve the interoperability problem . one solution would be to add personal patient characteristics such as family name , first name , date of birth ( separately hashed ) to be able to link data even in the case of field errors or other local errors to the ssn , which would help to comply with the recommendations of the international association of medical information technology , and where possible to ensure interoperability of this identifier with a european identifier . we propose to add to the european health card , as well as the national social insurance number of the patients in each country , a family - based identifier which could contribute to the harmonisation of patients ' identification at the european level . this solution would lead to the creation of a european health identifier which would allow patient data to be gathered anywhere in europe , whatever their location , even if their social insurance number changes according to their country of residence . this will be useful , at the individual level , to provide higher quality health care due to a better followup of the patient and to facilitate the reimbursement of health care costs . at the community level the great advantage of this european health identifier based on the family component is that it is built from very basic information , which is available to everybody , easily checkable and permanent throughout the patient life . our proposal to use the family - based identifier to create a unique european identifier will make it possible to link the data of a patient even when he or she resides outside his or her home country in europe . it will also contribute to the establishment of european public health statistics by matching healthcare data of the patients records with other administrative data ( mortality , social information , etc . ) after anonymisation of these data in accordance with the european directive on data protection . we could also propose to use biometric technologies ( and then apply hash coding to ensure anonymity ) . biometric technologies are sometimes proposed as a way to associate a patient with his or her medical data , as they do not require the patient to bring any documents or remember any information . though this technology represents real progress both in the identification and in the authentication of the patient , there are still many questions regarding the accuracy and reliability of each biometric technology and the associated costs . but the main problem lies in the acceptability of such systems by organisations concerned with ethical considerations such as patients ' associations , national ethics committees , human rights associations , and national committees for data protection . for example , in france , the use of biometric solutions for identification in the field of health has not been approved by the national ethics committee . initially , the government planned to use the ssn as an hin , as health professionals already know the ssn for health reimbursement reasons . patients ' associations , those defending individual liberties , national councils of medical associations , have asked for an identity number hin that is different from the ssn , their requirements are the following . first , that patients are made aware that the hin used for medical care is not the same as the ssn.second , that there are two different databases for health professionals ( located in hospitals or in other structures ) : a database for health reimbursements which will exclusively use the ssn for administrative reasons , and another medical care database for medical purposes which will not use the ssn because of its private aspect , but an anonymous identity number hin different from the ssn . we had to comply with these requirements and are thus obliged to provide tables to show the link between the ssn and the hin for all patients in all health structures for administrative purposes , which can be considered as the most important disadvantage of the proposed solution . first , that patients are made aware that the hin used for medical care is not the same as the ssn . second , that there are two different databases for health professionals ( located in hospitals or in other structures ) : a database for health reimbursements which will exclusively use the ssn for administrative reasons , and another medical care database for medical purposes which will not use the ssn because of its private aspect , but an anonymous identity number hin different from the ssn . patients ' associations and national councils of medical associations have been aware of the need for a correspondence table between administrative databases ( for health reimbursements ) and personal databases ( for medical purposes ) . this correspondence table was not a real problem for them as such tables currently exist in hospitals because of the need to link medical personal data ( hospital discharge abstracts referenced by the first and second name , date of birth , etc . ) with billing data referenced by the ssn . this particularly arises from the fact that administrators need to have access to medical information in order to determine the levels of hospital activity and justify hospital budgets . the solution for the existing correspondence table between administrative databases ( for health reimbursements ) and personal databases ( for medical purposes ) was to put it under the responsibility of the medical information department . that is why the associations concerned consider that using an hin obtained through irreversible transformation of the ssn provides more security than the current situation where the medical data are stored with personal characteristics ( first and second name , date of birth , etc . ) . we consider that our proposal must be accompanied by the reinforcement of measures regarding the security of medical information inside hospitals , as proposed at the end of the section 3 . in particular , we focused on the need to code ( reversible encryption ) both the hin and ssn in hospitals when they are related to health - care databases . as a consequence , the main concern regarding the personal medical record ( pmr ) and centralised storage of patient medical information is the security of data , not only at the level of the health structures , but also at the data - processing shelterer . knowing that a correspondence table between the ssn and the first hin would exist in health structures , we propose transforming the first hin into a storing hin by using an irreversible encryption method to secure storage ( data - processing shelterer ) . as only the manager of the portal will know the key of this second hashing , no dictionary attacks can be used to try to obtain the first hin , and thus the corresponding ssn through the correspondence table ( if hashed at the health structure level ) . first , in terms of organisation , it avoids implementation of the hashing function at the level of the health professional . second , it also avoids supplementary costs , implementation delays , and the disclosure of the technical hashing system to other actors . the second advantage is that it provides the possibility to use different hash - coding keys to generate a different hin for each major application such as the pmr , regional , and/or national applications such as health care networks , health administration , diagnosis and healthcare , and epidemiological research without delivering the corresponding application keys to all of the health professionals . moreover , hashing responsibility and key management can be delegated to each application . if the concept of specific hins for different applications is accepted , it will be necessary to create a structure similar to the data matching agency created in australia to manage all of the identifiers . this agency could be in charge of matching with regard to legal , deontological , organisational , and technical aspects , since the trustworthiness of such an agency is based on a complex combination of legal obligations ( in order to provide justifiable legal reliability ) and organisational - and - technical security measures ( in order to provide justifiable robustness with regard to accuracy of data ) . management of hins consists in not only ensuring the linkage of the different databases , but also guaranteeing the security of the different identifiers . for example , the real risks raised by using the same identifier in regional and national applications have to be considered : it is important to preclude the possibility of unauthorized linkage by indirect means . this agency could also manage the distribution of encryption keys among the different applications and actors . to ensure that legislation is respected and the interests of the patients protected , the agency would liaise with national commissions for data protection . our proposal for a french health identification number would make it possible to uniquely identify and link a patient to his or her specific medical data . hashing the social security number will allow linkage between the information of the personal medical file and other national sources of health information with the aim of completing or validating the personal medical record or performing epidemiological research . adding personal patient characteristics such as first name and family name , date of birth and/or biometric identifiers ( separately hashed , then merged ) to the hashed social security number could also be proposed in the discussions about the creation of a european health care identifier . it would thus contribute to the establishment of european public health statistics by matching healthcare data of the patients records with other administrative data . these data linkage systems would meet the requirements for anonymous data issued in the european directive on data protection .
we propose a method utilizing a derived social security number with the same reliability as the social security number . we show the anonymity techniques classically based on unidirectional hash functions ( such as the secure hash algorithm ( sha-2 ) function that can guarantee the security , quality , and reliability of information if these techniques are applied to the social security number ) . hashing produces a strictly anonymous code that is always the same for a given individual , and thus enables patient data to be linked . different solutions are developed and proposed in this article . hashing the social security number will make it possible to link the information in the personal medical file to other national health information sources with the aim of completing or validating the personal medical record or conducting epidemiological and clinical research . this data linkage would meet the anonymous data requirements of the european directive on data protection .
You are an expert at summarizing long articles. Proceed to summarize the following text: the fruit fly ( drosophila melanogaster ) compound eye is a great model system to investigate the functional organization of photoreceptor and interneuron arrays for neural image sampling and processing , and for animal vision . the system has the most complete wiring diagram and is amiable to genetic manipulations and accurate neural activity monitoring ( of high signal - to - noise ratio and time - resolution ) . the drosophila eye is modular , containing ~750 seemingly regular lens - capped structures called ommatidia , which together provide the fly a panoramic visual field that covers almost every direction around its head . each ommatidium contains eight photoreceptor cells ( r1-r8 ) , which share the same facet lens but are aligned to seven different directions . whilst the outer photoreceptors r1-r6 are most sensitive to blue - green light , spectral sensitivities of the inner cells r7 and r8 , which lie on top of each other and point to the same direction , exhibit three distinctive subtypes : pale , yellow and dorsal rim area ( dra ) . ( a ) the two first optic ganglia , retina and lamina , are highlighted in gray inside the fly eye . retina r1-r6 photoreceptors and lamina large monopolar cells ( lmcs : l1-l3 ) are readily accessible in vivo to conventional sharp microelectrode recordings . one path to record from lmcs in the lamina is to shift in parallel the electrode to left . ( b ) lamina is a matrix of retinotopically organized cartridges , each of which is packed with neurons that processes information from a specific small area in the visual space . due to neural superposition , six photoreceptors from different neighboring ommatidia send their axons ( r1-r6 ) to the same lamina cartridge , forming histaminergic output synapses to l1-l3 and an amacrine cell ( am ) . ( c ) the spread of neural information between r1-r6 axon terminals and the visual interneurons ( including l4 , l5 , lawf , c2 , c3 and t1 ) , inside a lamina cartridge is complex . ( d ) r1-r6 photoreceptor axons receive synaptic feedbacks from l2 and l4 monopolar cells . this means that the neural signals of eight photoreceptors belonging to seven neighboring ommatidia , which look at the same point in space , are pooled together at one neural cartridge in the next two neuropils : the lamina and medulla . while the six outer photoreceptors r1-r6 project their axon terminals to neural columns in the lamina ( figure 1 ) , r7 and r8 cells bypass this layer and make synaptic contacts with their corresponding medulla column . these exact wirings produce the neural substrate for the retinotopic mapping of fly early vision , whereupon every lamina ( figures 1a - c ) and medulla column ( cartridge ) represents a single point in space . direct inputs from r1-r6 photoreceptors are received by the large monopolar cells ( lmcs : l1 , l2 and l3 ) and the amacrine cell ( am ) in the lamina . out of these , l1 and l2 are the largest cells , mediating major information pathways ( figure 1d ) , which respond to on- and off - moving edges , and thus form the computational basis of the motion detector . behavioral experiments suggests that at intermediate contrast , the two pathways facilitate motion perception of opposite directions : back - to - front in l1 and front - to - back in l2 cells . connectivity further implies that l4 neurons may play critical role in the lateral communication between neighboring cartridges . reciprocal synapses were found between l2 and l4 cells located in the same and two adjacent cartridges . downstream , each l2 cell and its three associated l4 cells project their axons to a common target , the tm2 neuron in the medulla , where inputs from neighboring cartridges are believed to be integrated for processing of front - to - back motion . although l1 neurons receive input from same - cartridge l2s via both gap junctions and synapses , they are not directly connected to l4s and hence adjacent lamina cartridges . synaptic feedbacks to r1-r6 photoreceptor axons are provided only by neurons belonging to the l2/l4 circuits but not the l1 pathway ( figure 1d ) . whilst same - cartridge connections are selectively from l2 to r1 and r2 and from l4 to r5 , all r1-r6 photoreceptors receive synaptic feedback from l4 of either or both neighboring cartridges . furthermore , there are strong synaptic connections from am to r1 , r2 , r4 and r5 , and glia cells are also synaptically connected to the network and may thus participate in neural image processing . finally , axonal gap - junctions , linking neighboring r1-r6 and between r6 and r7/r8 photoreceptors in the lamina , contribute to the asymmetric information representation and processing in each cartridge . intracellular voltage recordings from individual photoreceptors and visual interneurons in nearly intact drosophila provide high signal - to - noise ratio data at sub - millisecond resolution , which is necessary for making sense of the fast neural computations between the connected neurons . this level of precision is impossible by current optical imaging techniques , which are significantly noisier and typically operate at 10 - 100 msec resolution . furthermore , because the electrodes have very small and sharp tips , the method is not restricted to cell bodies , but can provide direct recordings from small active neural structures ; such as the lmcs ' dendritic trees or photoreceptor axons , which can not be accessed by much larger tips of patch - clamp electrodes . importantly , the method is also structurally less invasive and damaging than most patch - clamp applications , and so affects less the studied cells ' intracellular milieu and information sampling . thus , conventional sharp microelectrode techniques have contributed , and keep on contributing , fundamental discoveries and original insight into neural information processing at the appropriate time scale ; improving our mechanistic understanding of vision . this article explains how in vivo intracellular recordings from drosophila r1-r6 photoreceptors and lmcs are performed in the juusola laboratory . this protocol will describe how to construct a suitable electrophysiology rig , prepare the fly , and perform the recordings . some representative data is presented , and some common issues and potential solutions are discussed that may be encountered when using this method . the following protocol complies with all the animal care guidelines of the university of sheffield and beijing normal university . recording and light stimulation equipment setup choose at least a 2.5 x 2.5 m recording area for performing electrophysiological experiments in a room that has air conditioning with regulated humidity and means to provide dark recording conditions . ensure that this area is large enough to comfortably fit a : ( i ) 1 x 1 m vibration - isolation table that houses the rig [ fly stimulation and recording apparatus ] , stereomicroscope and a cold light source with two goose necks , all enclosed within a large > 180 cm tall faraday cage ; ( ii ) a 38u equipment rack for housing a personal computer with a flat lcd monitor , microelectrode amplifier , led drivers , filters , temperature control units , oscilloscopes and other required electrical instruments ; and ( iii ) a small desk and a chair for the investigator.place the rig far away from electrical and mechanical noise sources , such as refrigerators , centrifuges and elevators . use separate surge protectors to protect the rig 's electrical devices from voltage spikes occurring in the mains . ideally , connect the rig to its own uninterruptible power supply ( ups battery ) to minimize noise.construct a conical fly - holder out of brass and black plastic ( figure 2 ) . drill a small tapering hole through the brass unit with its external rim narrowing to ~0.8 mm diameter ( matching a typical fly 's thorax width ) . note : this hole needs to taper toward the tip of the fly - holder so that a larger than average drosophila , which is projected from below by airflow , would get stuck shoulder - deep at the top rim.design and build a mechanically - robust , yet precise , fly stimulation and recording apparatus ( figure 3 ) . construct out of aluminum or brass ( high conductivity metals ) a fly preparation platform pole and around it a cardan - arm system , with embedded ball bearings , to provide smooth and accurate x , y - positioning and locking of the light stimulus . note : this integrated composite design minimizes mechanical vibrations , which could otherwise dislodge the recording electrode tip out from the studied cell . it can further incorporate a peltier - element - based close - loop temperature control system , enabling investigators to use temperature - sensitive genetic constructs , such as shibire , for assessing synaptic circuit computations . anodize the apparatus or paint it black to minimize light stimulus scatter . fix the fly stimulation and recording apparatus on the anti - vibration table 's breadboard ; for example by m6-bolts , using its metric screw holes . use a black breadboard or cover it with black fabric to minimize light scatter during experiments.position and lock ( using a locking screw ) a vertically adjustable fly preparation platform pole at center of a cardan - arm system . place the fly - preparation ( within the fly - holder , see step 2 ) on the platform pole so that the light source attached to the cardan - arm radially points to the fly 's head . ensure that the center of the fly eyes is exactly at the intersection point ( 0 , 0 ) of the cardan - arm 's x- and y - axes , as this enables accurate x , y - positioning of the light stimulus to any point within the fly 's visual field . note : this functionality is necessary for mapping the response properties of individual cells to specific eye locations ; e.g. , when searching for electrophysiological evidence for structural adaptations , such as bright or acute zones , which would show increasing sensitivity or resolution , respectively . mount the stereomicroscope behind the fly stimulation and recording apparatus on the anti - vibration table so that it provides comfortable high magnification viewing of the fly eye.mount the cold light source on the top of the microscope with the light source 's dual head semi - rigid gooseneck light guides pointing down toward the fly preparation holder . freely movable two beam illumination makes it easier to visualize the recording electrode tip when driving it through a small opening into the fly eye.attach an appropriate x , y , z - micromanipulator set ( coarse & fine ) for the recording electrode and the head - stage on the anti - vibration table , at the right side of the fly stimulation and recording apparatus , using m6-bolts or magnetic stands . note : in the juusola laboratory , different rigs are equipped with different manipulators ; for details see the table of materials and reagents . these all provide high - quality intracellular recordings.mount a small manual 3-axis micromanipulator for the reference electrode holder on the vertically adjustable fly preparation platform pole . orient the reference electrode so that it is pointing toward the fly preparation.construct a free - standing light - shielded faraday cage out of steel - panels around the anti - vibration table , surrounding the fly stimulation and recording apparatus , to prevent outside electromagnetic interference . leave the front of the cage open , providing access to transport the fly preparation for the experiments . attach black fabric curtains ( having copper- or aluminum - mesh implanted inside them for grounding ) at the front to shield out noise and light . paint the interior of the cage black to minimize light scatter and bolt the feet of the cage on the floor to prevent vibrations.connect the voltage and current outputs of the high - impedance intracellular microelectrode amplifier to the inputs of two separate low - pass filters ( bessel or similar ) using bnc - cables . likewise , connect the filter outputs into the appropriate channels of the ad - connector blocks / boards of the data acquisition system ( da / ad cards ) . connect the da / ad card(s ) into a personal computer by specialized cables , according to supplier manuals.install appropriate acquisition software for the data acquisition system of choice on the personal computer . ensure that the data acquisition drivers are compatible with the operating system on the personal computer.ground electrically the fly stimulation and recording apparatus , faraday cage , copper mesh ( within the curtains ) , microscope , micromanipulators , cold light source , 38u equipment rack with all its instruments ( the intracellular amplifier , filters , temperature control unit , pc and lcd monitor etc . ) to a single central ground point by using equipment grounding wire and m6 crimp ring grounding ends . use an electric multimeter to test that all the parts are in the same ground . note : to achieve the best possible low - noise recording conditions , the grounding configurations typically vary from one set - up to another . if needed , connect the central ground point further to the building ground , and/or the microelectrode amplifier 's central ground . after testing the fully functioning system during real electrophysiological experiments , be prepared to change the grounding configuration as needed to minimize noise in the recordings . configure software amplification ( 1 - 10x ) , signal filtering ( typically low - pass filters set at 500 hz , which is suitable for both r1-r6 and lmc data ) , and sampling rate ( at least 1 khz ) . ensure that the settings obey nyquist - shannon sampling theorem ; for example , when acquiring data that is low - pass - filtered at 500 hz , use a sampling frequency of 1 khz or higher to minimize aliasing effects . as characteristic voltage responses of r1-r6 photoreceptors are 40 - 65 mv , and those of lmcs 20 - 45 mv , set the amplification and display scales accordingly to enable high - resolution sampling and data visualization . choose at least a 2.5 x 2.5 m recording area for performing electrophysiological experiments in a room that has air conditioning with regulated humidity and means to provide dark recording conditions . ensure that this area is large enough to comfortably fit a : ( i ) 1 x 1 m vibration - isolation table that houses the rig [ fly stimulation and recording apparatus ] , stereomicroscope and a cold light source with two goose necks , all enclosed within a large > 180 cm tall faraday cage ; ( ii ) a 38u equipment rack for housing a personal computer with a flat lcd monitor , microelectrode amplifier , led drivers , filters , temperature control units , oscilloscopes and other required electrical instruments ; and ( iii ) a small desk and a chair for the investigator . place the rig far away from electrical and mechanical noise sources , such as refrigerators , centrifuges and elevators . use separate surge protectors to protect the rig 's electrical devices from voltage spikes occurring in the mains . ideally , connect the rig to its own uninterruptible power supply ( ups battery ) to minimize noise . construct a conical fly - holder out of brass and black plastic ( figure 2 ) . drill a small tapering hole through the brass unit with its external rim narrowing to ~0.8 mm diameter ( matching a typical fly 's thorax width ) . note : this hole needs to taper toward the tip of the fly - holder so that a larger than average drosophila , which is projected from below by airflow , would get stuck shoulder - deep at the top rim . design and build a mechanically - robust , yet precise , fly stimulation and recording apparatus ( figure 3 ) . construct out of aluminum or brass ( high conductivity metals ) a fly preparation platform pole and around it a cardan - arm system , with embedded ball bearings , to provide smooth and accurate x , y - positioning and locking of the light stimulus . note : this integrated composite design minimizes mechanical vibrations , which could otherwise dislodge the recording electrode tip out from the studied cell . it can further incorporate a peltier - element - based close - loop temperature control system , enabling investigators to use temperature - sensitive genetic constructs , such as shibire , for assessing synaptic circuit computations . fix the fly stimulation and recording apparatus on the anti - vibration table 's breadboard ; for example by m6-bolts , using its metric screw holes . use a black breadboard or cover it with black fabric to minimize light scatter during experiments.position and lock ( using a locking screw ) a vertically adjustable fly preparation platform pole at center of a cardan - arm system . place the fly - preparation ( within the fly - holder , see step 2 ) on the platform pole so that the light source attached to the cardan - arm radially points to the fly 's head . ensure that the center of the fly eyes is exactly at the intersection point ( 0 , 0 ) of the cardan - arm 's x- and y - axes , as this enables accurate x , y - positioning of the light stimulus to any point within the fly 's visual field . note : this functionality is necessary for mapping the response properties of individual cells to specific eye locations ; e.g. , when searching for electrophysiological evidence for structural adaptations , such as bright or acute zones , which would show increasing sensitivity or resolution , respectively . fix the fly stimulation and recording apparatus on the anti - vibration table 's breadboard ; for example by m6-bolts , using its metric screw holes . use a black breadboard or cover it with black fabric to minimize light scatter during experiments . position and lock ( using a locking screw ) a vertically adjustable fly preparation platform pole at center of a cardan - arm system . place the fly - preparation ( within the fly - holder , see step 2 ) on the platform pole so that the light source attached to the cardan - arm radially points to the fly 's head . ensure that the center of the fly eyes is exactly at the intersection point ( 0 , 0 ) of the cardan - arm 's x- and y - axes , as this enables accurate x , y - positioning of the light stimulus to any point within the fly 's visual field . note : this functionality is necessary for mapping the response properties of individual cells to specific eye locations ; e.g. , when searching for electrophysiological evidence for structural adaptations , such as bright or acute zones , which would show increasing sensitivity or resolution , respectively . mount the stereomicroscope behind the fly stimulation and recording apparatus on the anti - vibration table so that it provides comfortable high magnification viewing of the fly eye . mount the cold light source on the top of the microscope with the light source 's dual head semi - rigid gooseneck light guides pointing down toward the fly preparation holder . freely movable two beam illumination makes it easier to visualize the recording electrode tip when driving it through a small opening into the fly eye . attach an appropriate x , y , z - micromanipulator set ( coarse & fine ) for the recording electrode and the head - stage on the anti - vibration table , at the right side of the fly stimulation and recording apparatus , using m6-bolts or magnetic stands . note : in the juusola laboratory , different rigs are equipped with different manipulators ; for details see the table of materials and reagents . mount a small manual 3-axis micromanipulator for the reference electrode holder on the vertically adjustable fly preparation platform pole . construct a free - standing light - shielded faraday cage out of steel - panels around the anti - vibration table , surrounding the fly stimulation and recording apparatus , to prevent outside electromagnetic interference . leave the front of the cage open , providing access to transport the fly preparation for the experiments . attach black fabric curtains ( having copper- or aluminum - mesh implanted inside them for grounding ) at the front to shield out noise and light . paint the interior of the cage black to minimize light scatter and bolt the feet of the cage on the floor to prevent vibrations . connect the voltage and current outputs of the high - impedance intracellular microelectrode amplifier to the inputs of two separate low - pass filters ( bessel or similar ) using bnc - cables . likewise , connect the filter outputs into the appropriate channels of the ad - connector blocks / boards of the data acquisition system ( da / ad cards ) . connect the da / ad card(s ) into a personal computer by specialized cables , according to supplier manuals . install appropriate acquisition software for the data acquisition system of choice on the personal computer . ensure that the data acquisition drivers are compatible with the operating system on the personal computer . ground electrically the fly stimulation and recording apparatus , faraday cage , copper mesh ( within the curtains ) , microscope , micromanipulators , cold light source , 38u equipment rack with all its instruments ( the intracellular amplifier , filters , temperature control unit , pc and lcd monitor etc . ) to a single central ground point by using equipment grounding wire and m6 crimp ring grounding ends . use an electric multimeter to test that all the parts are in the same ground . note : to achieve the best possible low - noise recording conditions , the grounding configurations typically vary from one set - up to another . if needed , connect the central ground point further to the building ground , and/or the microelectrode amplifier 's central ground . after testing the fully functioning system during real electrophysiological experiments , be prepared to change the grounding configuration as needed to minimize noise in the recordings . if needed , connect the central ground point further to the building ground , and/or the microelectrode amplifier 's central ground . after testing the fully functioning system during real electrophysiological experiments , be prepared to change the grounding configuration as needed to minimize noise in the recordings . configure software amplification ( 1 - 10x ) , signal filtering ( typically low - pass filters set at 500 hz , which is suitable for both r1-r6 and lmc data ) , and sampling rate ( at least 1 khz ) . ensure that the settings obey nyquist - shannon sampling theorem ; for example , when acquiring data that is low - pass - filtered at 500 hz , use a sampling frequency of 1 khz or higher to minimize aliasing effects . as characteristic voltage responses of r1-r6 photoreceptors are 40 - 65 mv , and those of lmcs 20 - 45 mv , set the amplification and display scales accordingly to enable high - resolution sampling and data visualization . as characteristic voltage responses of r1-r6 photoreceptors are 40 - 65 mv , and those of lmcs 20 - 45 mv , set the amplification and display scales accordingly to enable high - resolution sampling and data visualization . the fly - holder is made out of two pieces : the central brass unit and its conical black plastic coat . the central hole inside the brass unit tapers to a small diameter that barely lets the fly through . the set - up contains a free - standing light - shielded faraday cage , the anti - vibration table , the fly stimulation and recording apparatus , and black fabric curtains with copper- or aluminum - mesh inside for grounding . the instrument rack is electrically connected to the same central ground with all the equipment inside the faraday cage . fabricating microelectrodes pull the reference microelectrode from filamented borosilicate ( outer diameter : 1.0 mm ; inner diameter 0.6 mm ) or quartz glass ( outer diameter : 1.0 mm ; inner diameter 0.5 , 0.6 or 0.7 mm ) tubing using a pipette puller instrument . try to achieve a short gradual taper . note : the exact settings of the pipette puller program vary from instrument to instrument ; more details in the table of materials and regents . the pore size at the tip is not crucial because the tip of the reference electrode will be broken before being inserted into the fly preparation.pull the recording microelectrode from filamented borosilicate ( outer diameter : 1.0 mm ; inner diameter 0.6 mm ) or quartz glass ( outer diameter : 1.0 mm ; inner diameter 0.5 , 0.6 or 0.7 mm ) tubing using a pipette puller instrument . try to achieve a long ( 10 - 15 mm ) fine gradual taper.inspect with a light microscope that the recording electrodes show correct tapering . mount the electrode on a glass slide with moldable adhesive and use 40x air objective to inspect its tip . note : a good electrode tapers smoothly until its invisibly small tip , around which continuous parallel darker and lighter interference patterns can be seen . some puller settings generate high resistance electrodes , which can not yield successful cell penetrations because their tips resemble " trumpets " . thus , visual inspection of the electrodes is important.attach the electrodes horizontally on a large petri - dish with modelling clay ( or similar moldable adhesive ) for safe - keeping and transportation to the electrophysiology rig . ensure that the electrode tips are always in the air and not accidentally touching anything.back-fill the recording and reference electrodes just before the experiment with the appropriate salt solution . use a small 5 ml syringe connected to a small particle filter with a fine plastic tip ( such as a microloader ) . for photoreceptors experiments , fill the recording electrode until full ( a droplet forms in its large end ) with 3 m kcl as this solution minimizes the effect of liquid junction potential to the recorded voltage.for investigating the histaminergic lmcs , which respond to synaptic input from r1-r6 photoreceptors by chloride - conductance changes , fill the recording electrodes with 3 m potassium acetate and 0.5 mm kcl , as this solution has less effect on the cell 's chloride battery . fill the reference electrode with fly ringer , containing in mm : 120 nacl , 5 kcl , 10 tes ( c6h15no6s ) , 1.5 cacl2 , 4 mgcl2 , and 30 sucrose . ensure that the silver wires inside the electrode - holders are evenly coated with silver chloride ( appearing purple - grey - not shiny silvery ) to minimize recording artefacts ( such as drift in the junction potential ) . if not , replace them with properly chloridized wires.if needed , chloridize new silver wires . carefully clean the wires ( by quickly passing them through a flame ) so that these appear bright silver in color . avoid touching them with the fingers , in order to deposit on an even layer of agcl . soak the wires in full strength household bleach for 15 - 30 min until they appear purple - gray color . alternatively , electroplate each wire ( by making it positive with respect to a solution containing 3 m kcl and passing a current through it at a rate of 1 ma / cm of surface area ) for 10 - 15 sec until adequately coated.connect the back - filled recording and reference electrodes to their electrode - holders . place a small ringer 's solution bath on the vertically adjustable fly preparation platform pole . drive the electrode tips into the ringer 's solution and measure the recording electrode 's tip resistance . note : this step is only needed when testing the resistive properties of electrodes , which are pulled from a new batch of glass tubing , or when optimizing the microelectrode puller instrument programs through iteration.before performing the resistance measurements , read the instructions in the amplifier manufacture 's user manual for the appropriate measurement settings . for a good recording electrode , have a tip resistance of ~100 - 220 m. pull the reference microelectrode from filamented borosilicate ( outer diameter : 1.0 mm ; inner diameter 0.6 mm ) or quartz glass ( outer diameter : 1.0 mm ; inner diameter 0.5 , 0.6 or 0.7 mm ) tubing using a pipette puller instrument . note : the exact settings of the pipette puller program vary from instrument to instrument ; more details in the table of materials and regents . the pore size at the tip is not crucial because the tip of the reference electrode will be broken before being inserted into the fly preparation . pull the recording microelectrode from filamented borosilicate ( outer diameter : 1.0 mm ; inner diameter 0.6 mm ) or quartz glass ( outer diameter : 1.0 mm ; inner diameter 0.5 , 0.6 or 0.7 mm ) tubing using a pipette puller instrument . mount the electrode on a glass slide with moldable adhesive and use 40x air objective to inspect its tip . note : a good electrode tapers smoothly until its invisibly small tip , around which continuous parallel darker and lighter interference patterns can be seen . some puller settings generate high resistance electrodes , which can not yield successful cell penetrations because their tips resemble " trumpets " . attach the electrodes horizontally on a large petri - dish with modelling clay ( or similar moldable adhesive ) for safe - keeping and transportation to the electrophysiology rig . ensure that the electrode tips are always in the air and not accidentally touching anything . back - fill the recording and reference electrodes just before the experiment with the appropriate salt solution . use a small 5 ml syringe connected to a small particle filter with a fine plastic tip ( such as a microloader ) . for photoreceptors experiments , fill the recording electrode until full ( a droplet forms in its large end ) with 3 m kcl as this solution minimizes the effect of liquid junction potential to the recorded voltage.for investigating the histaminergic lmcs , which respond to synaptic input from r1-r6 photoreceptors by chloride - conductance changes , fill the recording electrodes with 3 m potassium acetate and 0.5 mm kcl , as this solution has less effect on the cell 's chloride battery . fill the reference electrode with fly ringer , containing in mm : 120 nacl , 5 kcl , 10 tes ( c6h15no6s ) , 1.5 cacl2 , 4 mgcl2 , and 30 sucrose . for photoreceptors experiments , fill the recording electrode until full ( a droplet forms in its large end ) with 3 m kcl as this solution minimizes the effect of liquid junction potential to the recorded voltage . for investigating the histaminergic lmcs , which respond to synaptic input from r1-r6 photoreceptors by chloride - conductance changes , fill the recording electrodes with 3 m potassium acetate and 0.5 mm kcl , as this solution has less effect on the cell 's chloride battery . fill the reference electrode with fly ringer , containing in mm : 120 nacl , 5 kcl , 10 tes ( c6h15no6s ) , 1.5 cacl2 , 4 mgcl2 , and 30 sucrose . ensure that the silver wires inside the electrode - holders are evenly coated with silver chloride ( appearing purple - grey - not shiny silvery ) to minimize recording artefacts ( such as drift in the junction potential ) . if not , replace them with properly chloridized wires.if needed , chloridize new silver wires . carefully clean the wires ( by quickly passing them through a flame ) so that these appear bright silver in color . avoid touching them with the fingers , in order to deposit on an even layer of agcl . soak the wires in full strength household bleach for 15 - 30 min until they appear purple - gray color . alternatively , electroplate each wire ( by making it positive with respect to a solution containing 3 m kcl and passing a current through it at a rate of 1 ma / cm of surface area ) for 10 - 15 sec until adequately coated.connect the back - filled recording and reference electrodes to their electrode - holders . place a small ringer 's solution bath on the vertically adjustable fly preparation platform pole . drive the electrode tips into the ringer 's solution and measure the recording electrode 's tip resistance . note : this step is only needed when testing the resistive properties of electrodes , which are pulled from a new batch of glass tubing , or when optimizing the microelectrode puller instrument programs through iteration.before performing the resistance measurements , read the instructions in the amplifier manufacture 's user manual for the appropriate measurement settings . for a good recording electrode , have a tip resistance of ~100 - 220 m. ensure that the silver wires inside the electrode - holders are evenly coated with silver chloride ( appearing purple - grey - not shiny silvery ) to minimize recording artefacts ( such as drift in the junction potential ) . if not , replace them with properly chloridized wires . carefully clean the wires ( by quickly passing them through a flame ) so that these appear bright silver in color . avoid touching them with the fingers , in order to deposit on an even layer of agcl . soak the wires in full strength household bleach for 15 - 30 min until they appear purple - gray color . alternatively , electroplate each wire ( by making it positive with respect to a solution containing 3 m kcl and passing a current through it at a rate of 1 ma / cm of surface area ) for 10 - 15 sec until adequately coated . connect the back - filled recording and reference electrodes to their electrode - holders . place a small ringer 's solution bath on the vertically adjustable fly preparation platform pole . drive the electrode tips into the ringer 's solution and measure the recording electrode 's tip resistance . note : this step is only needed when testing the resistive properties of electrodes , which are pulled from a new batch of glass tubing , or when optimizing the microelectrode puller instrument programs through iteration . before performing the resistance measurements , read the instructions in the amplifier manufacture 's user manual for the appropriate measurement settings . for a good recording electrode , have a tip resistance of ~100 - 220 m. collect 5 - 10 days old flies ( after eclosion ) and place them in a clean fly tube containing standard food . it is possible to achieve good recordings from younger flies too , even from the " newborns " ; but because of their softer eyes , cutting a corneal opening for the recording electrode is more difficult . construct a fly catching tube and a fly preparation stand ( figure 4 ) . see figure 4 for the general idea of how these self - made tools were put together . to make a fly catching tube , cut off the tip of 50 ml plastic centrifuge tube 's conical bottom . then , insert and glue the large end of 1 ml pipette tip on this new opening.finally , cut the small end of pipette to a size that readily lets a fly to walk through . consult a mechanical workshop to assemble a small fly preparation stage that enables 2-axes rotation and locking of the fly - holder to different positions . to make a fly catching tube , cut off the tip of 50 ml plastic centrifuge tube 's conical bottom . then , insert and glue the large end of 1 ml pipette tip on this new opening . finally , cut the small end of pipette to a size that readily lets a fly to walk through . consult a mechanical workshop to assemble a small fly preparation stage that enables 2-axes rotation and locking of the fly - holder to different positions . fly catching tube is made by gluing a 1 ml plastic pipette tip to a 50 ml plastic centrifuge tube . bespoke fly preparation stand enables free - rotation and locking of the fly - holder in a preferred position for preparing the fly . petroleum jelly is applied by a small applicator made by connecting a thick sort hair on a handle . collect a fly for the experiment in a 1 ml pipette tip , which barely enables a fly to fit through . attach the fly catching tube , with the pipette tip on it , to the fly tube . in catching a fly , take advantage of their inherent tendency to climb upwards ( antigravitaxis ) into the pipette tip . note : the bigger the fly , the bigger its cells and the better the chances for high quality intracellular recordings . smaller flies ( both females and males ) can also provide excellent recordings , but the preparation is more difficult to make . once the fly is trapped in a large pipette tip , remember to close the fly tube to stop other flies from escaping . connect a 100 ml syringe with a flexible plastic hose to the larger opening of the pipette tip - with the fly still in it . place the narrow end on the large pipette tip , which is enlarged just to let a drosophila through , to the opening in the bottom of the fly - holder and squeeze a small volume of air from the syringe to eject the fly into the fly - holder . look through the stereomicroscope and gently administer more air until the fly 's head is protruding from the conical end of the fly - holder . ensure that the fly is firmly trapped from its thorax to the small opening on the top of the fly - holder . look through the stereomicroscope and gently administer more air until the fly 's head is protruding from the conical end of the fly - holder . ensure that the fly is firmly trapped from its thorax to the small opening on the top of the fly - holder . use a wax heater to fasten the fly with beeswax from its " shoulders " to the fly - holder . adjust the temperature of the wax heater to be as low as possible yet cleanly melting the wax . note : when the temperature is correct , the wax appears transparent . too high of a temperature makes the wax " burn away " ; too low keeps the wax stiff . when fixing the fly , be accurate and brief as prolonged heat exposure may damage it . using light - cured dental glue . left , a drosophila 's head is positioned straight in the fly - holder and fixed from its proboscis , right eye and shoulders to the fly - holder with heated beeswax . right , a small opening is cut in the thickest part of the eye , just above the equator and only a few ommatidia away from the back cuticle , using a sharp razor edge . a piece of cornea is gently removed and the hole is sealed with petroleum jelly to prevent the eye from drying up . apply beeswax to the proboscis ( figure 5 ) and the corner of the right eye , avoiding the cornea , and fix the head from these points to the fly - holder . produce a micro - knife . clamp a non - stainless - steel razorblade with two blade - holders / breakers ( both with flat grip ) and crack a small strip of its sharp edge . for health and safety , use goggles for eye protection ( even though it is highly unlikely that pieces would ricochet when the razor is cracked ) . ideally , produce a sharp razor edge that resembles a spire . ensure that this " spire " is firmly attached to the blade - holder , but be careful to avoid any self - injury ! using the micro - knife , prepare a small opening size of few ommatidia in the fly 's left eye - at about 4 - 5 ommatidia from the dorsal cuticle just above the eye 's equator to provide the passage for the recording microelectrode . note : because the fly eye feels elastic and resistive to probing , the hole is best cut with a " spire "- knife . the cutting technique is quite challenging , so pay close attention to the video demonstration . keeping the fly - holder in certain orientations ( in the fly preparation stand ) can make the dissection easier . initially , the microsurgery may feel difficult to learn , but once committed , neural adaptation gradually improves the investigator 's 3d - perception and dexterity . remove carefully the small piece of cornea from the opening that was just cut , exposing the retina underneath . swiftly cover the hole in the eye with a tiny blob of petroleum jelly using the fine hair of the petroleum jelly applicator . it prevents tissue dehydration and coagulation of the hemolymph that would break the inserted recording electrode . this can improve the frequency response of the recording system , and so the temporal resolution of the recorded neural signals . avoid smearing petroleum jelly over the rest of the eye as this blurs the optics . always be grounded when operating the microelectrode amplifier ( for example by touching the metal surface of the faraday cage or anti - vibration table ) , as this precludes one from accidentally delivering a static charge to the head - stage , which could damage the circuitry . illuminate the fly preparation platform pole from above by two goose - neck light guides ( figure 6a ) ( with the cold light source inside the faraday cage ) so that the fly - holder can be placed on the pole in the preferred position under close visual control . ( a - b ) the fly - holder is placed on the recording platform that also provides temperature control via a peltier element ( a : white round platform in the center ) . the cardan - arm enables exact positioning of the light stimulus at an equal distance ( via x , y - rotation ) around the fly , with the light source ( a liquid or quartz fiber - optic bundle end ) directly pointing to its eye . in many of our rigs , light stimulation is generated by leds ( with linear current - drivers ) or by a monochromator . thus , their stimuli carry a specific ( band - passed ) spectral content , selected between 300 - 740 nm and cover 4 - 6 log intensity unit range ( as attenuated by separate neutral density filters ) . ( c ) two microelectrodes , controlled by separate micromanipulators , are positioned in the fly head : the reference electrode ( above ) through the ocelli ; the recording electrode ( left ) through the small opening in the left eye . ( d ) for obtaining a maximum number of photoreceptor recordings , the recording microelectrode is driven into the hole , parallel to the proboscis - ocellus axis . when the electrode tip penetrates and seals to a photoreceptor , the freely rotatable light source is fixed to the position where the cell produces the maximum voltage response to a given light stimulus . if the hole is close to the cuticle , lmc penetrations can further be achieved with this same electrode angle ( left ) . if the hole is further from the cuticle , another useful electrode approach angle to obtain lmc recordings is also shown ( right ) . mount the fly - holder ( with the fly in it ! ) on the fly preparation platform pole . rotate the fly - holder so that the fly 's left eye is directly facing the investigator ( figure 6b ) . insert the blunt reference electrode gently through the fly 's occelli into the head capsule using a small coarse micromanipulator while observing the preparation through the stereomicroscope ( figure 6c ) . do not push the electrode too deep , as this can damage the fly brain . alternatively , insert the reference electrode into the back of the thorax . always , ensure that the fly appears heathy ( moves its antennae ) and its eyes are intact ; not accidentally damaged . if the preparation looks less than immaculate , prepare a new fly for the experiments . always , ensure that the fly appears heathy ( moves its antennae ) and its eyes are intact ; not accidentally damaged . if the preparation looks less than immaculate , prepare a new fly for the experiments . drive the sharp recording microelectrode into the left eye through the petroleum jelly covered small opening prepared earlier . use high magnification in the stereomicroscope and move the light guides and the focal plane so that the electrode tip location becomes apparent in 3d by its reflectance patterns . note : figure 6d shows how the fly 's head should be positioned slightly differently ( in respect to the angle the recording microelectrode enters the eye ) for photoreceptor and lmc recordings . driving the electrode into the eye without breaking it is the most difficult phase of the experiment . if the electrode tip misses the small opening in the eye , hitting the cornea , it typically breaks . turn on the microelectrode amplifier once both electrodes are firmly inside the preparation , in electrical contact with the fly 's body fluids . turn off the cold - light source ( inside the faraday cage ) and unplug it from the mains . connect its plug to the central ground to minimize ground - loop induced electrical noise , and move the goose - neck light guides away so that the cardan - arm system can be freely moved around the fly . switch off the room lights to ensure that the fly preparation is now in relative darkness . measure the resistance of the recording electrode in the eye ( as instructed in the amplifier 's user manual ) . use only recording electrodes in which resistance is 100 - 250 m. note : it is virtually impossible to achieve high quality intracellular recordings by < 70 m electrode . if the resistance is < 80 m , it is likely that the electrode tip is broken . in this case , switch off the amplifier and change the recording electrode . once the electrode is replaced and in the eye , switch on the amplifier to measure its resistance . sometimes , the electrode tip can become blocked by some detritus as it enters the tissue . this can be remedied by using the amplifier 's capacitive buzz and current pulse functions that typically clear it by rapid resonation or repulsion . once the electrode is replaced and in the eye , switch on the amplifier to measure its resistance . sometimes , the electrode tip can become blocked by some detritus as it enters the tissue . this can be remedied by using the amplifier 's capacitive buzz and current pulse functions that typically clear it by rapid resonation or repulsion . cancel out any arbitrary voltage difference between the recording and reference electrodes , as both of them are now resting in the electrically interconnected extracellular space , by setting the signal offset ( recording voltage ) to zero . follow the signal offset changes using the amplifier 's display readout or an oscilloscope screen . drive the recording electrode tip gradually deeper into the eye with small 0.1 to 1 micron steps . do this with an x - axis piezo - stepper of a remote - controlled micromanipulator or by gently rotating the fine resolution knob of a manual manipulator . stimulate the fly eye with brief ( 1 - 10 msec ) light flashes as the recording electrode is being advanced in the tissue . note : if the recording electrode is positioned in the retina and the eye functions normally , each light flash will cause a brief and small drop in the voltage ( 0.2 - 5 mv hyperpolarization ) , called the electroretinogram ( erg ) . this change in the field potential of the extracellular space is caused by the retinal cells ' collective response to light . however , once the electrode tip enters the lamina , closing on the lmcs , the erg reverses , showing depolarizing responses . move the light source around the fly eye by using the cardan - arm system and find the position where the light evokes the largest erg response . note : this position marks the small area in the visual space where the photoreceptors ( or lmcs ) , which are located next to the tip of the recording electrode , sample their light input . note : the penetration can occur spontaneously , or when the electrode is micro - stepped forward . it can be further facilitated by gently tapping the micromanipulator system or by using the buzz - function of the amplifier ; these actions resonate the electrode tip in the tissue . when the electrode impales the photoreceptor membrane , entering its intracellular space , the voltage difference between the recording and reference electrode drops suddenly from 0 mv to ~-65 mv ( between -55 and -75 mv ) ; whereas during lmc penetrations , this drop is typically less ( between -30 and -50 mv ) . these voltage differences represent the negative resting potentials of the given cells . depending on the quality of the recording electrode ( its sharpness ) and the cellular process it penetrated , the voltage reading from the recording electrode can stabilize rapidly or gradually to the resting potential , as the cell membrane seals to the outer layer of the electrode . but if the penetration is only partial or poor , the electrode typically slips out of the cell with the recorded potential climbing back towards zero . localize the center of the penetrated cell 's receptive field when the electrode appears properly sealed , showing stable membrane potential ( dark resting potential ) . move the flashing light stimulus around the fly eye , using the cardan - arm system , to find the point in visual space , where the light flash evokes the cell 's maximum voltage response . lock the cardan - arm when the light stimulus directly faces ( points at ) the receptive field center . note : in darkness , drosophila photoreceptors respond to bright light pulses with 40 - 65 mv depolarizing voltage responses , while stable lmc recordings show 20 to 45 mv hyperpolarizing responses . -80 mv resting potentials and much slower and smaller ( ~5 mv ) saturated light - induced depolarizations . photoreceptors in drosophila with different eye pigmentations , such as white - eye and cinnabar , show comparable response sizes to wild - type . using the amplifier 's current - clamp ( cc ) mode , compensate the recording electrode 's capacitance by injecting small 0.1 na and brief ( 100 - 200 msec ) current pulses into the studied cell to minimize recording artefacts during its membrane charging . note : this important procedure is explained in detail in the amplifier 's user manual , and should be practiced with an electrical cell model before the actual experiments . record voltage responses to light pulses and other stimuli of interest , having varying statistical or physical qualities ( such as naturalistic light intensity time series or random contrast patterns ) . test , for example , how the recorded responses change with light- or dark - adaptation . note : one can accurately light - adapt the studied cell by continuous light of preselected intensity by adding neutral density filters on the light path . alternatively , for prolonged dark - adaptation switch off the light stimulus for a preset time . because of the mechanical stability of the recording system , the high quality of the recording electrodes and the intactness of the preparation thus , on a good day , it is possible to collect a large amount of data at different adapting conditions from a single cell . when the electrode slips out of the cell , the recorded responses diminish and the mean voltage begins to approach zero . advance carefully the recording electrode with the fine x - axis control of the micromanipulator until the electrode makes contact and penetrates the next cell ( this is typically the nearest neural neighbor ) . do not move the electrode along y- or z - axis as these maneuvers would make the electrode to " plough the tissue " sideways , damaging eye structures ! note : with a good electrode and a healthy preparation , one can record high quality responses from many photoreceptors ( but rarely from many lmcs ) in the same fly over a period of several hours ; occasionally , over the whole working day ( > 8 hr ) without clear signal deterioration . save data files periodically with identifying information , such as date , genotype , and the recorded cell type . because of the large amount of data that can be collected in a successful recording session , keep good written records in a lab - book for future data analysis . recording and light stimulation equipment setup choose at least a 2.5 x 2.5 m recording area for performing electrophysiological experiments in a room that has air conditioning with regulated humidity and means to provide dark recording conditions . ensure that this area is large enough to comfortably fit a : ( i ) 1 x 1 m vibration - isolation table that houses the rig [ fly stimulation and recording apparatus ] , stereomicroscope and a cold light source with two goose necks , all enclosed within a large > 180 cm tall faraday cage ; ( ii ) a 38u equipment rack for housing a personal computer with a flat lcd monitor , microelectrode amplifier , led drivers , filters , temperature control units , oscilloscopes and other required electrical instruments ; and ( iii ) a small desk and a chair for the investigator.place the rig far away from electrical and mechanical noise sources , such as refrigerators , centrifuges and elevators . use separate surge protectors to protect the rig 's electrical devices from voltage spikes occurring in the mains . ideally , connect the rig to its own uninterruptible power supply ( ups battery ) to minimize noise.construct a conical fly - holder out of brass and black plastic ( figure 2 ) . drill a small tapering hole through the brass unit with its external rim narrowing to ~0.8 mm diameter ( matching a typical fly 's thorax width ) . note : this hole needs to taper toward the tip of the fly - holder so that a larger than average drosophila , which is projected from below by airflow , would get stuck shoulder - deep at the top rim.design and build a mechanically - robust , yet precise , fly stimulation and recording apparatus ( figure 3 ) . construct out of aluminum or brass ( high conductivity metals ) a fly preparation platform pole and around it a cardan - arm system , with embedded ball bearings , to provide smooth and accurate x , y - positioning and locking of the light stimulus . note : this integrated composite design minimizes mechanical vibrations , which could otherwise dislodge the recording electrode tip out from the studied cell . it can further incorporate a peltier - element - based close - loop temperature control system , enabling investigators to use temperature - sensitive genetic constructs , such as shibire , for assessing synaptic circuit computations . fix the fly stimulation and recording apparatus on the anti - vibration table 's breadboard ; for example by m6-bolts , using its metric screw holes . use a black breadboard or cover it with black fabric to minimize light scatter during experiments.position and lock ( using a locking screw ) a vertically adjustable fly preparation platform pole at center of a cardan - arm system . place the fly - preparation ( within the fly - holder , see step 2 ) on the platform pole so that the light source attached to the cardan - arm radially points to the fly 's head . ensure that the center of the fly eyes is exactly at the intersection point ( 0 , 0 ) of the cardan - arm 's x- and y - axes , as this enables accurate x , y - positioning of the light stimulus to any point within the fly 's visual field . note : this functionality is necessary for mapping the response properties of individual cells to specific eye locations ; e.g. , when searching for electrophysiological evidence for structural adaptations , such as bright or acute zones , which would show increasing sensitivity or resolution , respectively . mount the stereomicroscope behind the fly stimulation and recording apparatus on the anti - vibration table so that it provides comfortable high magnification viewing of the fly eye.mount the cold light source on the top of the microscope with the light source 's dual head semi - rigid gooseneck light guides pointing down toward the fly preparation holder . freely movable two beam illumination makes it easier to visualize the recording electrode tip when driving it through a small opening into the fly eye.attach an appropriate x , y , z - micromanipulator set ( coarse & fine ) for the recording electrode and the head - stage on the anti - vibration table , at the right side of the fly stimulation and recording apparatus , using m6-bolts or magnetic stands . note : in the juusola laboratory , different rigs are equipped with different manipulators ; for details see the table of materials and reagents . these all provide high - quality intracellular recordings.mount a small manual 3-axis micromanipulator for the reference electrode holder on the vertically adjustable fly preparation platform pole . orient the reference electrode so that it is pointing toward the fly preparation.construct a free - standing light - shielded faraday cage out of steel - panels around the anti - vibration table , surrounding the fly stimulation and recording apparatus , to prevent outside electromagnetic interference . leave the front of the cage open , providing access to transport the fly preparation for the experiments . attach black fabric curtains ( having copper- or aluminum - mesh implanted inside them for grounding ) at the front to shield out noise and light . paint the interior of the cage black to minimize light scatter and bolt the feet of the cage on the floor to prevent vibrations.connect the voltage and current outputs of the high - impedance intracellular microelectrode amplifier to the inputs of two separate low - pass filters ( bessel or similar ) using bnc - cables . likewise , connect the filter outputs into the appropriate channels of the ad - connector blocks / boards of the data acquisition system ( da / ad cards ) . connect the da / ad card(s ) into a personal computer by specialized cables , according to supplier manuals.install appropriate acquisition software for the data acquisition system of choice on the personal computer . ensure that the data acquisition drivers are compatible with the operating system on the personal computer.ground electrically the fly stimulation and recording apparatus , faraday cage , copper mesh ( within the curtains ) , microscope , micromanipulators , cold light source , 38u equipment rack with all its instruments ( the intracellular amplifier , filters , temperature control unit , pc and lcd monitor etc . ) to a single central ground point by using equipment grounding wire and m6 crimp ring grounding ends . use an electric multimeter to test that all the parts are in the same ground . note : to achieve the best possible low - noise recording conditions , the grounding configurations typically vary from one set - up to another . if needed , connect the central ground point further to the building ground , and/or the microelectrode amplifier 's central ground . after testing the fully functioning system during real electrophysiological experiments , be prepared to change the grounding configuration as needed to minimize noise in the recordings . configure software amplification ( 1 - 10x ) , signal filtering ( typically low - pass filters set at 500 hz , which is suitable for both r1-r6 and lmc data ) , and sampling rate ( at least 1 khz ) . ensure that the settings obey nyquist - shannon sampling theorem ; for example , when acquiring data that is low - pass - filtered at 500 hz , use a sampling frequency of 1 khz or higher to minimize aliasing effects . as characteristic voltage responses of r1-r6 photoreceptors are 40 - 65 mv , and those of lmcs 20 - 45 mv , set the amplification and display scales accordingly to enable high - resolution sampling and data visualization . choose at least a 2.5 x 2.5 m recording area for performing electrophysiological experiments in a room that has air conditioning with regulated humidity and means to provide dark recording conditions . ensure that this area is large enough to comfortably fit a : ( i ) 1 x 1 m vibration - isolation table that houses the rig [ fly stimulation and recording apparatus ] , stereomicroscope and a cold light source with two goose necks , all enclosed within a large > 180 cm tall faraday cage ; ( ii ) a 38u equipment rack for housing a personal computer with a flat lcd monitor , microelectrode amplifier , led drivers , filters , temperature control units , oscilloscopes and other required electrical instruments ; and ( iii ) a small desk and a chair for the investigator . place the rig far away from electrical and mechanical noise sources , such as refrigerators , centrifuges and elevators . use separate surge protectors to protect the rig 's electrical devices from voltage spikes occurring in the mains . ideally , connect the rig to its own uninterruptible power supply ( ups battery ) to minimize noise . construct a conical fly - holder out of brass and black plastic ( figure 2 ) . drill a small tapering hole through the brass unit with its external rim narrowing to ~0.8 mm diameter ( matching a typical fly 's thorax width ) . note : this hole needs to taper toward the tip of the fly - holder so that a larger than average drosophila , which is projected from below by airflow , would get stuck shoulder - deep at the top rim . design and build a mechanically - robust , yet precise , fly stimulation and recording apparatus ( figure 3 ) . construct out of aluminum or brass ( high conductivity metals ) a fly preparation platform pole and around it a cardan - arm system , with embedded ball bearings , to provide smooth and accurate x , y - positioning and locking of the light stimulus . note : this integrated composite design minimizes mechanical vibrations , which could otherwise dislodge the recording electrode tip out from the studied cell . it can further incorporate a peltier - element - based close - loop temperature control system , enabling investigators to use temperature - sensitive genetic constructs , such as shibire , for assessing synaptic circuit computations . fix the fly stimulation and recording apparatus on the anti - vibration table 's breadboard ; for example by m6-bolts , using its metric screw holes . use a black breadboard or cover it with black fabric to minimize light scatter during experiments.position and lock ( using a locking screw ) a vertically adjustable fly preparation platform pole at center of a cardan - arm system . place the fly - preparation ( within the fly - holder , see step 2 ) on the platform pole so that the light source attached to the cardan - arm radially points to the fly 's head . ensure that the center of the fly eyes is exactly at the intersection point ( 0 , 0 ) of the cardan - arm 's x- and y - axes , as this enables accurate x , y - positioning of the light stimulus to any point within the fly 's visual field . note : this functionality is necessary for mapping the response properties of individual cells to specific eye locations ; e.g. , when searching for electrophysiological evidence for structural adaptations , such as bright or acute zones , which would show increasing sensitivity or resolution , respectively . fix the fly stimulation and recording apparatus on the anti - vibration table 's breadboard ; for example by m6-bolts , using its metric screw holes . use a black breadboard or cover it with black fabric to minimize light scatter during experiments . position and lock ( using a locking screw ) a vertically adjustable fly preparation platform pole at center of a cardan - arm system . place the fly - preparation ( within the fly - holder , see step 2 ) on the platform pole so that the light source attached to the cardan - arm radially points to the fly 's head . ensure that the center of the fly eyes is exactly at the intersection point ( 0 , 0 ) of the cardan - arm 's x- and y - axes , as this enables accurate x , y - positioning of the light stimulus to any point within the fly 's visual field . note : this functionality is necessary for mapping the response properties of individual cells to specific eye locations ; e.g. , when searching for electrophysiological evidence for structural adaptations , such as bright or acute zones , which would show increasing sensitivity or resolution , respectively . mount the stereomicroscope behind the fly stimulation and recording apparatus on the anti - vibration table so that it provides comfortable high magnification viewing of the fly eye . mount the cold light source on the top of the microscope with the light source 's dual head semi - rigid gooseneck light guides pointing down toward the fly preparation holder . freely movable two beam illumination makes it easier to visualize the recording electrode tip when driving it through a small opening into the fly eye . attach an appropriate x , y , z - micromanipulator set ( coarse & fine ) for the recording electrode and the head - stage on the anti - vibration table , at the right side of the fly stimulation and recording apparatus , using m6-bolts or magnetic stands . note : in the juusola laboratory , different rigs are equipped with different manipulators ; for details see the table of materials and reagents . mount a small manual 3-axis micromanipulator for the reference electrode holder on the vertically adjustable fly preparation platform pole . construct a free - standing light - shielded faraday cage out of steel - panels around the anti - vibration table , surrounding the fly stimulation and recording apparatus , to prevent outside electromagnetic interference . leave the front of the cage open , providing access to transport the fly preparation for the experiments . attach black fabric curtains ( having copper- or aluminum - mesh implanted inside them for grounding ) at the front to shield out noise and light . paint the interior of the cage black to minimize light scatter and bolt the feet of the cage on the floor to prevent vibrations . connect the voltage and current outputs of the high - impedance intracellular microelectrode amplifier to the inputs of two separate low - pass filters ( bessel or similar ) using bnc - cables . likewise , connect the filter outputs into the appropriate channels of the ad - connector blocks / boards of the data acquisition system ( da / ad cards ) . connect the da / ad card(s ) into a personal computer by specialized cables , according to supplier manuals . install appropriate acquisition software for the data acquisition system of choice on the personal computer . ensure that the data acquisition drivers are compatible with the operating system on the personal computer . ground electrically the fly stimulation and recording apparatus , faraday cage , copper mesh ( within the curtains ) , microscope , micromanipulators , cold light source , 38u equipment rack with all its instruments ( the intracellular amplifier , filters , temperature control unit , pc and lcd monitor etc . ) to a single central ground point by using equipment grounding wire and m6 crimp ring grounding ends . use an electric multimeter to test that all the parts are in the same ground . note : to achieve the best possible low - noise recording conditions , the grounding configurations typically vary from one set - up to another . if needed , connect the central ground point further to the building ground , and/or the microelectrode amplifier 's central ground . after testing the fully functioning system during real electrophysiological experiments , be prepared to change the grounding configuration as needed to minimize noise in the recordings . if needed , connect the central ground point further to the building ground , and/or the microelectrode amplifier 's central ground . after testing the fully functioning system during real electrophysiological experiments , be prepared to change the grounding configuration as needed to minimize noise in the recordings . configure software amplification ( 1 - 10x ) , signal filtering ( typically low - pass filters set at 500 hz , which is suitable for both r1-r6 and lmc data ) , and sampling rate ( at least 1 khz ) . ensure that the settings obey nyquist - shannon sampling theorem ; for example , when acquiring data that is low - pass - filtered at 500 hz , use a sampling frequency of 1 khz or higher to minimize aliasing effects . as characteristic voltage responses of r1-r6 photoreceptors are 40 - 65 mv , and those of lmcs 20 - 45 mv , set the amplification and display scales accordingly to enable high - resolution sampling and data visualization . as characteristic voltage responses of r1-r6 photoreceptors are 40 - 65 mv , and those of lmcs 20 - 45 mv , set the amplification and display scales accordingly to enable high - resolution sampling and data visualization . the fly - holder is made out of two pieces : the central brass unit and its conical black plastic coat . the central hole inside the brass unit tapers to a small diameter that barely lets the fly through . the set - up contains a free - standing light - shielded faraday cage , the anti - vibration table , the fly stimulation and recording apparatus , and black fabric curtains with copper- or aluminum - mesh inside for grounding . the instrument rack is electrically connected to the same central ground with all the equipment inside the faraday cage . fabricating microelectrodes pull the reference microelectrode from filamented borosilicate ( outer diameter : 1.0 mm ; inner diameter 0.6 mm ) or quartz glass ( outer diameter : 1.0 mm ; inner diameter 0.5 , 0.6 or 0.7 mm ) tubing using a pipette puller instrument . note : the exact settings of the pipette puller program vary from instrument to instrument ; more details in the table of materials and regents . the pore size at the tip is not crucial because the tip of the reference electrode will be broken before being inserted into the fly preparation.pull the recording microelectrode from filamented borosilicate ( outer diameter : 1.0 mm ; inner diameter 0.6 mm ) or quartz glass ( outer diameter : 1.0 mm ; inner diameter 0.5 , 0.6 or 0.7 mm ) tubing using a pipette puller instrument . try to achieve a long ( 10 - 15 mm ) fine gradual taper.inspect with a light microscope that the recording electrodes show correct tapering . mount the electrode on a glass slide with moldable adhesive and use 40x air objective to inspect its tip . note : a good electrode tapers smoothly until its invisibly small tip , around which continuous parallel darker and lighter interference patterns can be seen . some puller settings generate high resistance electrodes , which can not yield successful cell penetrations because their tips resemble " trumpets " . thus , visual inspection of the electrodes is important.attach the electrodes horizontally on a large petri - dish with modelling clay ( or similar moldable adhesive ) for safe - keeping and transportation to the electrophysiology rig . ensure that the electrode tips are always in the air and not accidentally touching anything.back-fill the recording and reference electrodes just before the experiment with the appropriate salt solution . use a small 5 ml syringe connected to a small particle filter with a fine plastic tip ( such as a microloader ) . for photoreceptors experiments , fill the recording electrode until full ( a droplet forms in its large end ) with 3 m kcl as this solution minimizes the effect of liquid junction potential to the recorded voltage.for investigating the histaminergic lmcs , which respond to synaptic input from r1-r6 photoreceptors by chloride - conductance changes , fill the recording electrodes with 3 m potassium acetate and 0.5 mm kcl , as this solution has less effect on the cell 's chloride battery . fill the reference electrode with fly ringer , containing in mm : 120 nacl , 5 kcl , 10 tes ( c6h15no6s ) , 1.5 cacl2 , 4 mgcl2 , and 30 sucrose . ensure that the silver wires inside the electrode - holders are evenly coated with silver chloride ( appearing purple - grey - not shiny silvery ) to minimize recording artefacts ( such as drift in the junction potential ) . if not , replace them with properly chloridized wires.if needed , chloridize new silver wires . carefully clean the wires ( by quickly passing them through a flame ) so that these appear bright silver in color . avoid touching them with the fingers , in order to deposit on an even layer of agcl . soak the wires in full strength household bleach for 15 - 30 min until they appear purple - gray color . alternatively , electroplate each wire ( by making it positive with respect to a solution containing 3 m kcl and passing a current through it at a rate of 1 ma / cm of surface area ) for 10 - 15 sec until adequately coated.connect the back - filled recording and reference electrodes to their electrode - holders . place a small ringer 's solution bath on the vertically adjustable fly preparation platform pole . drive the electrode tips into the ringer 's solution and measure the recording electrode 's tip resistance . note : this step is only needed when testing the resistive properties of electrodes , which are pulled from a new batch of glass tubing , or when optimizing the microelectrode puller instrument programs through iteration.before performing the resistance measurements , read the instructions in the amplifier manufacture 's user manual for the appropriate measurement settings . for a good recording electrode , have a tip resistance of ~100 - 220 m. pull the reference microelectrode from filamented borosilicate ( outer diameter : 1.0 mm ; inner diameter 0.6 mm ) or quartz glass ( outer diameter : 1.0 mm ; inner diameter 0.5 , 0.6 or 0.7 mm ) tubing using a pipette puller instrument . note : the exact settings of the pipette puller program vary from instrument to instrument ; more details in the table of materials and regents . the pore size at the tip is not crucial because the tip of the reference electrode will be broken before being inserted into the fly preparation . pull the recording microelectrode from filamented borosilicate ( outer diameter : 1.0 mm ; inner diameter 0.6 mm ) or quartz glass ( outer diameter : 1.0 mm ; inner diameter 0.5 , 0.6 or 0.7 mm ) tubing using a pipette puller instrument . mount the electrode on a glass slide with moldable adhesive and use 40x air objective to inspect its tip . note : a good electrode tapers smoothly until its invisibly small tip , around which continuous parallel darker and lighter interference patterns can be seen . some puller settings generate high resistance electrodes , which can not yield successful cell penetrations because their tips resemble " trumpets " . attach the electrodes horizontally on a large petri - dish with modelling clay ( or similar moldable adhesive ) for safe - keeping and transportation to the electrophysiology rig . ensure that the electrode tips are always in the air and not accidentally touching anything . back - fill the recording and reference electrodes just before the experiment with the appropriate salt solution . use a small 5 ml syringe connected to a small particle filter with a fine plastic tip ( such as a microloader ) . for photoreceptors experiments , fill the recording electrode until full ( a droplet forms in its large end ) with 3 m kcl as this solution minimizes the effect of liquid junction potential to the recorded voltage.for investigating the histaminergic lmcs , which respond to synaptic input from r1-r6 photoreceptors by chloride - conductance changes , fill the recording electrodes with 3 m potassium acetate and 0.5 mm kcl , as this solution has less effect on the cell 's chloride battery . fill the reference electrode with fly ringer , containing in mm : 120 nacl , 5 kcl , 10 tes ( c6h15no6s ) , 1.5 cacl2 , 4 mgcl2 , and 30 sucrose . for photoreceptors experiments , fill the recording electrode until full ( a droplet forms in its large end ) with 3 m kcl as this solution minimizes the effect of liquid junction potential to the recorded voltage . for investigating the histaminergic lmcs , which respond to synaptic input from r1-r6 photoreceptors by chloride - conductance changes , fill the recording electrodes with 3 m potassium acetate and 0.5 mm kcl , as this solution has less effect on the cell 's chloride battery . fill the reference electrode with fly ringer , containing in mm : 120 nacl , 5 kcl , 10 tes ( c6h15no6s ) , 1.5 cacl2 , 4 mgcl2 , and 30 sucrose . ensure that the silver wires inside the electrode - holders are evenly coated with silver chloride ( appearing purple - grey - not shiny silvery ) to minimize recording artefacts ( such as drift in the junction potential ) . if not , replace them with properly chloridized wires.if needed , chloridize new silver wires . carefully clean the wires ( by quickly passing them through a flame ) so that these appear bright silver in color . avoid touching them with the fingers , in order to deposit on an even layer of agcl . soak the wires in full strength household bleach for 15 - 30 min until they appear purple - gray color . alternatively , electroplate each wire ( by making it positive with respect to a solution containing 3 m kcl and passing a current through it at a rate of 1 ma / cm of surface area ) for 10 - 15 sec until adequately coated.connect the back - filled recording and reference electrodes to their electrode - holders . place a small ringer 's solution bath on the vertically adjustable fly preparation platform pole . drive the electrode tips into the ringer 's solution and measure the recording electrode 's tip resistance . note : this step is only needed when testing the resistive properties of electrodes , which are pulled from a new batch of glass tubing , or when optimizing the microelectrode puller instrument programs through iteration.before performing the resistance measurements , read the instructions in the amplifier manufacture 's user manual for the appropriate measurement settings . for a good recording electrode , have a tip resistance of ~100 - 220 m. ensure that the silver wires inside the electrode - holders are evenly coated with silver chloride ( appearing purple - grey - not shiny silvery ) to minimize recording artefacts ( such as drift in the junction potential ) . if not , replace them with properly chloridized wires . carefully clean the wires ( by quickly passing them through a flame ) so that these appear bright silver in color . avoid touching them with the fingers , in order to deposit on an even layer of agcl . soak the wires in full strength household bleach for 15 - 30 min until they appear purple - gray color . alternatively , electroplate each wire ( by making it positive with respect to a solution containing 3 m kcl and passing a current through it at a rate of 1 ma / cm of surface area ) for 10 - 15 sec until adequately coated . connect the back - filled recording and reference electrodes to their electrode - holders . place a small ringer 's solution bath on the vertically adjustable fly preparation platform pole . drive the electrode tips into the ringer 's solution and measure the recording electrode 's tip resistance . note : this step is only needed when testing the resistive properties of electrodes , which are pulled from a new batch of glass tubing , or when optimizing the microelectrode puller instrument programs through iteration . before performing the resistance measurements , read the instructions in the amplifier manufacture 's user manual for the appropriate measurement settings . collect 5 - 10 days old flies ( after eclosion ) and place them in a clean fly tube containing standard food . it is possible to achieve good recordings from younger flies too , even from the " newborns " ; but because of their softer eyes , cutting a corneal opening for the recording electrode is more difficult . construct a fly catching tube and a fly preparation stand ( figure 4 ) . see figure 4 for the general idea of how these self - made tools were put together . to make a fly catching tube , cut off the tip of 50 ml plastic centrifuge tube 's conical bottom . then , insert and glue the large end of 1 ml pipette tip on this new opening.finally , cut the small end of pipette to a size that readily lets a fly to walk through . consult a mechanical workshop to assemble a small fly preparation stage that enables 2-axes rotation and locking of the fly - holder to different positions . to make a fly catching tube , cut off the tip of 50 ml plastic centrifuge tube 's conical bottom . then , insert and glue the large end of 1 ml pipette tip on this new opening . finally , cut the small end of pipette to a size that readily lets a fly to walk through . consult a mechanical workshop to assemble a small fly preparation stage that enables 2-axes rotation and locking of the fly - holder to different positions . fly catching tube is made by gluing a 1 ml plastic pipette tip to a 50 ml plastic centrifuge tube . bespoke fly preparation stand enables free - rotation and locking of the fly - holder in a preferred position for preparing the fly . petroleum jelly is applied by a small applicator made by connecting a thick sort hair on a handle . collect a fly for the experiment in a 1 ml pipette tip , which barely enables a fly to fit through . attach the fly catching tube , with the pipette tip on it , to the fly tube . in catching a fly , take advantage of their inherent tendency to climb upwards ( antigravitaxis ) into the pipette tip . note : the bigger the fly , the bigger its cells and the better the chances for high quality intracellular recordings . smaller flies ( both females and males ) can also provide excellent recordings , but the preparation is more difficult to make . once the fly is trapped in a large pipette tip , remember to close the fly tube to stop other flies from escaping . connect a 100 ml syringe with a flexible plastic hose to the larger opening of the pipette tip - with the fly still in it . place the narrow end on the large pipette tip , which is enlarged just to let a drosophila through , to the opening in the bottom of the fly - holder and squeeze a small volume of air from the syringe to eject the fly into the fly - holder . look through the stereomicroscope and gently administer more air until the fly 's head is protruding from the conical end of the fly - holder . ensure that the fly is firmly trapped from its thorax to the small opening on the top of the fly - holder . look through the stereomicroscope and gently administer more air until the fly 's head is protruding from the conical end of the fly - holder . ensure that the fly is firmly trapped from its thorax to the small opening on the top of the fly - holder . use a wax heater to fasten the fly with beeswax from its " shoulders " to the fly - holder . adjust the temperature of the wax heater to be as low as possible yet cleanly melting the wax . too high of a temperature makes the wax " burn away " ; too low keeps the wax stiff . when fixing the fly , be accurate and brief as prolonged heat exposure may damage it . using light - cured dental glue . left , a drosophila 's head is positioned straight in the fly - holder and fixed from its proboscis , right eye and shoulders to the fly - holder with heated beeswax . right , a small opening is cut in the thickest part of the eye , just above the equator and only a few ommatidia away from the back cuticle , using a sharp razor edge . a piece of cornea is gently removed and the hole is sealed with petroleum jelly to prevent the eye from drying up . apply beeswax to the proboscis ( figure 5 ) and the corner of the right eye , avoiding the cornea , and fix the head from these points to the fly - holder . produce a micro - knife . clamp a non - stainless - steel razorblade with two blade - holders / breakers ( both with flat grip ) and use goggles for eye protection ( even though it is highly unlikely that pieces would ricochet when the razor is cracked ) . ideally , produce a sharp razor edge that resembles a spire . ensure that this " spire " is firmly attached to the blade - holder , but be careful to avoid any self - injury ! using the micro - knife , prepare a small opening size of few ommatidia in the fly 's left eye - at about 4 - 5 ommatidia from the dorsal cuticle just above the eye 's equator to provide the passage for the recording microelectrode . note : because the fly eye feels elastic and resistive to probing , the hole is best cut with a " spire "- knife . the cutting technique is quite challenging , so pay close attention to the video demonstration . keeping the fly - holder in certain orientations ( in the fly preparation stand ) can make the dissection easier . initially , the microsurgery may feel difficult to learn , but once committed , neural adaptation gradually improves the investigator 's 3d - perception and dexterity . remove carefully the small piece of cornea from the opening that was just cut , exposing the retina underneath . swiftly cover the hole in the eye with a tiny blob of petroleum jelly using the fine hair of the petroleum jelly applicator . it prevents tissue dehydration and coagulation of the hemolymph that would break the inserted recording electrode . this can improve the frequency response of the recording system , and so the temporal resolution of the recorded neural signals . avoid smearing petroleum jelly over the rest of the eye as this blurs the optics . always be grounded when operating the microelectrode amplifier ( for example by touching the metal surface of the faraday cage or anti - vibration table ) , as this precludes one from accidentally delivering a static charge to the head - stage , which could damage the circuitry . illuminate the fly preparation platform pole from above by two goose - neck light guides ( figure 6a ) ( with the cold light source inside the faraday cage ) so that the fly - holder can be placed on the pole in the preferred position under close visual control . ( a - b ) the fly - holder is placed on the recording platform that also provides temperature control via a peltier element ( a : white round platform in the center ) . the cardan - arm enables exact positioning of the light stimulus at an equal distance ( via x , y - rotation ) around the fly , with the light source ( a liquid or quartz fiber - optic bundle end ) directly pointing to its eye . in many of our rigs , light stimulation is generated by leds ( with linear current - drivers ) or by a monochromator . thus , their stimuli carry a specific ( band - passed ) spectral content , selected between 300 - 740 nm and cover 4 - 6 log intensity unit range ( as attenuated by separate neutral density filters ) . ( c ) two microelectrodes , controlled by separate micromanipulators , are positioned in the fly head : the reference electrode ( above ) through the ocelli ; the recording electrode ( left ) through the small opening in the left eye . ( d ) for obtaining a maximum number of photoreceptor recordings , the recording microelectrode is driven into the hole , parallel to the proboscis - ocellus axis . when the electrode tip penetrates and seals to a photoreceptor , the freely rotatable light source is fixed to the position where the cell produces the maximum voltage response to a given light stimulus . if the hole is close to the cuticle , lmc penetrations can further be achieved with this same electrode angle ( left ) . if the hole is further from the cuticle , another useful electrode approach angle to obtain lmc recordings is also shown ( right ) . please click here to view a larger version of this figure . mount the fly - holder ( with the fly in it ! ) on the fly preparation platform pole . rotate the fly - holder so that the fly 's left eye is directly facing the investigator ( figure 6b ) . insert the blunt reference electrode gently through the fly 's occelli into the head capsule using a small coarse micromanipulator while observing the preparation through the stereomicroscope ( figure 6c ) . do not push the electrode too deep , as this can damage the fly brain . alternatively , insert the reference electrode into the back of the thorax . always , ensure that the fly appears heathy ( moves its antennae ) and its eyes are intact ; not accidentally damaged . if the preparation looks less than immaculate , prepare a new fly for the experiments . ensure that the fly appears heathy ( moves its antennae ) and its eyes are intact ; not accidentally damaged . if the preparation looks less than immaculate , prepare a new fly for the experiments . drive the sharp recording microelectrode into the left eye through the petroleum jelly covered small opening prepared earlier . use high magnification in the stereomicroscope and move the light guides and the focal plane so that the electrode tip location becomes apparent in 3d by its reflectance patterns . note : figure 6d shows how the fly 's head should be positioned slightly differently ( in respect to the angle the recording microelectrode enters the eye ) for photoreceptor and lmc recordings . driving the electrode into the eye without breaking it is the most difficult phase of the experiment . if the electrode tip misses the small opening in the eye , hitting the cornea , it typically breaks . turn on the microelectrode amplifier once both electrodes are firmly inside the preparation , in electrical contact with the fly 's body fluids . turn off the cold - light source ( inside the faraday cage ) and unplug it from the mains . connect its plug to the central ground to minimize ground - loop induced electrical noise , and move the goose - neck light guides away so that the cardan - arm system can be freely moved around the fly . switch off the room lights to ensure that the fly preparation is now in relative darkness . measure the resistance of the recording electrode in the eye ( as instructed in the amplifier 's user manual ) . use only recording electrodes in which resistance is 100 - 250 m. note : it is virtually impossible to achieve high quality intracellular recordings by < 70 m electrode . if the resistance is < 80 m , it is likely that the electrode tip is broken . in this case , switch off the amplifier and change the recording electrode . once the electrode is replaced and in the eye , switch on the amplifier to measure its resistance . sometimes , the electrode tip can become blocked by some detritus as it enters the tissue . this can be remedied by using the amplifier 's capacitive buzz and current pulse functions that typically clear it by rapid resonation or repulsion . once the electrode is replaced and in the eye , switch on the amplifier to measure its resistance . sometimes , the electrode tip can become blocked by some detritus as it enters the tissue . this can be remedied by using the amplifier 's capacitive buzz and current pulse functions that typically clear it by rapid resonation or repulsion . cancel out any arbitrary voltage difference between the recording and reference electrodes , as both of them are now resting in the electrically interconnected extracellular space , by setting the signal offset ( recording voltage ) to zero . follow the signal offset changes using the amplifier 's display readout or an oscilloscope screen . drive the recording electrode tip gradually deeper into the eye with small 0.1 to 1 micron steps . do this with an x - axis piezo - stepper of a remote - controlled micromanipulator or by gently rotating the fine resolution knob of a manual manipulator . stimulate the fly eye with brief ( 1 - 10 msec ) light flashes as the recording electrode is being advanced in the tissue . note : if the recording electrode is positioned in the retina and the eye functions normally , each light flash will cause a brief and small drop in the voltage ( 0.2 - 5 mv hyperpolarization ) , called the electroretinogram ( erg ) . this change in the field potential of the extracellular space is caused by the retinal cells ' collective response to light . however , once the electrode tip enters the lamina , closing on the lmcs , the erg reverses , showing depolarizing responses . move the light source around the fly eye by using the cardan - arm system and find the position where the light evokes the largest erg response . note : this position marks the small area in the visual space where the photoreceptors ( or lmcs ) , which are located next to the tip of the recording electrode , sample their light input . note : the penetration can occur spontaneously , or when the electrode is micro - stepped forward . it can be further facilitated by gently tapping the micromanipulator system or by using the buzz - function of the amplifier ; these actions resonate the electrode tip in the tissue . when the electrode impales the photoreceptor membrane , entering its intracellular space , the voltage difference between the recording and reference electrode drops suddenly from 0 mv to ~-65 mv ( between -55 and -75 mv ) ; whereas during lmc penetrations , this drop is typically less ( between -30 and -50 mv ) . depending on the quality of the recording electrode ( its sharpness ) and the cellular process it penetrated , the voltage reading from the recording electrode can stabilize rapidly or gradually to the resting potential , as the cell membrane seals to the outer layer of the electrode . but if the penetration is only partial or poor , the electrode typically slips out of the cell with the recorded potential climbing back towards zero . localize the center of the penetrated cell 's receptive field when the electrode appears properly sealed , showing stable membrane potential ( dark resting potential ) . move the flashing light stimulus around the fly eye , using the cardan - arm system , to find the point in visual space , where the light flash evokes the cell 's maximum voltage response . lock the cardan - arm when the light stimulus directly faces ( points at ) the receptive field center . note : in darkness , drosophila photoreceptors respond to bright light pulses with 40 - 65 mv depolarizing voltage responses , while stable lmc recordings show 20 to 45 mv hyperpolarizing responses . -80 mv resting potentials and much slower and smaller ( ~5 mv ) saturated light - induced depolarizations . photoreceptors in drosophila with different eye pigmentations , such as white - eye and cinnabar , show comparable response sizes to wild - type . using the amplifier 's current - clamp ( cc ) mode , compensate the recording electrode 's capacitance by injecting small 0.1 na and brief ( 100 - 200 msec ) current pulses into the studied cell to minimize recording artefacts during its membrane charging . note : this important procedure is explained in detail in the amplifier 's user manual , and should be practiced with an electrical cell model before the actual experiments . record voltage responses to light pulses and other stimuli of interest , having varying statistical or physical qualities ( such as naturalistic light intensity time series or random contrast patterns ) . test , for example , how the recorded responses change with light- or dark - adaptation . note : one can accurately light - adapt the studied cell by continuous light of preselected intensity by adding neutral density filters on the light path . alternatively , for prolonged dark - adaptation switch off the light stimulus for a preset time . because of the mechanical stability of the recording system , the high quality of the recording electrodes and the intactness of the preparation thus , on a good day , it is possible to collect a large amount of data at different adapting conditions from a single cell . when the electrode slips out of the cell , the recorded responses diminish and the mean voltage begins to approach zero . advance carefully the recording electrode with the fine x - axis control of the micromanipulator until the electrode makes contact and penetrates the next cell ( this is typically the nearest neural neighbor ) . do not move the electrode along y- or z - axis as these maneuvers would make the electrode to " plough the tissue " sideways , damaging eye structures ! note : with a good electrode and a healthy preparation , one can record high quality responses from many photoreceptors ( but rarely from many lmcs ) in the same fly over a period of several hours ; occasionally , over the whole working day ( > 8 hr ) without clear signal deterioration . save data files periodically with identifying information , such as date , genotype , and the recorded cell type . because of the large amount of data that can be collected in a successful recording session , keep good written records in a lab - book for future data analysis . the sharp microelectrode recording method , as adapted here for the drosophila eye , can be used to reliably quantify neural information sampling and processing in the retina and lamina cells , and communication between them . by using it to study encoding in different wild - type stocks , mutants or genetically engineered fly strains , the method has proven its value ; not only in quantifying the effects of a mutation , temperature , diet or selected expression , but also in revealing mechanistic reasons for altered visual behaviors . the method is also readily applicable to other insect preparations , empowering neuroethological vision studies . figure 7.voltage responses of a fruit fly r1-r6 photoreceptor to a light pulse at 20 and 25 c. because the sharp microelectrode penetrations are often very stable , it is possible to record voltage responses of the same r1-r6 photoreceptor to a given light stimulus at different ambient temperatures by warming or cooling the fly . in our set - ups , the fly - holder is placed on a close - loop peltier - element - based temperature - control system . higher temperature accelerates the voltage responses and characteristically lowers the resting potential of r1-r6 photoreceptors ( as indicated by red arrows ) . please click here to view a larger version of this figure . studying the effect of temperature on photoreceptor output with a well - designed and vibration - isolated recording system , the method can be used for measuring the effect of temperature on an individual cell 's neural output by warming or cooling the fly . the given example shows voltage responses to a bright 10 msec long pulse , recorded in the same r1-r6 photoreceptor at 20 and 25 c ( figure 7 ) . as quantified before , warming lowers a photoreceptor 's resting potential in darkness , and accelerates its voltage responses . ( a ) photoreceptor output to dim ( below ) and bright ( above ; 10,000-times brighter light ) repeated naturalistic light intensity time series recorded by the same microelectrode in the same cell at 20 c. responses to the bright stimulus are larger , because they integrate more samples , elementary responses ( bumps ) to single photons . individual responses ( light gray ) were taken after the adaptive trends ( arrow in a ) had receded ( dotted box in a ) . ( c ) the cells ' signaling performance was quantified by the recordings ' signal - to - noise ratios ( snr ) using the standard methods . photoreceptor output has about 64 hz broader range of reliable signaling at the bright stimulation ( snr'bright 1 , up to 84 hz ) than at the dim ( snr'dim 1 , up to 20 hz ) , with signal - to - noise ratio improving greatly ; from snrdim= 87 to snrbright= 1,868 . studying adaptation and neural encoding by repetitive stimulation the noninvasiveness of the method , causing relatively little damage in the retina and lamina structures , makes it ideal for studying the signaling performance of individual cells to different light stimuli in their near natural physiological state in vivo . figure 8 shows voltage responses of a r1-r6 photoreceptor to a dim and bright repeated naturalistic light intensity time series stimulus at 20 c , whereas figure 9 shows responses of another r1-r6 photoreceptor and a lmc to a different naturalistic stimulus at 25 c. the pre- and postsynaptic recordings were performed separately from two different flies because simultaneous intracellular recordings by two sharp microelectrodes in the same fly , one in the retina and the other in the lamina , are too difficult to be viable . figure 9.voltage responses of a fruit fly r1-r6 photoreceptor and lmc to repeated naturalistic stimulation at 25 c. ( a ) r1-r6 ( gray ) and lmc ( black ) outputs recorded by different microelectrodes from different flies . ( b ) fully light - adapted 20 consecutive pre- ( above ) and postsynaptic ( below ) responses to the same naturalistic stimulus pattern with individual responses , shown in light gray and the corresponding response means ( the signals ) as the darker traces . ( c ) the cells ' signaling performance was quantified by the recordings ' signal - to - noise ratios ( snr ) . lmc output has about 10 hz broader range of reliable signaling ( snr'lmc 1 , up to 104 hz ) than r1-r6 output ( snr'r 1 , up to 94 hz ) . both signal - to - noise ratios are high ( snrlmc= 142 , snrr= 752 ) , and as the recording noise was low , their differences reflect real encoding differences between the cells after the stimulus onset , the recordings typically show fast adapting trends that largely subside within 5 - 6 sec . from then on , the cells produce highly consistent responses to each 1 sec long stimulus presentation ( each dotted box encloses 20 of these ) . the repeatability of the responses becomes obvious when these are superimposed ( figure 8b and figure 9b ) . individual responses are the thin gray traces , and their mean the thicker darker trace . the mean response is taken as the neural signal , whereas the neural noise is the difference between the mean and each individual response . the respective signal - to - noise ratios in frequency domain ( figure 8c and figure 9c ) were obtained by fourier - transforming the signal and noise data chunks into power spectra , and dividing the mean signal power spectrum with the corresponding mean noise power spectrum . characteristically , the maximum signal - to - noise ratios of the recorded neural outputs to naturalistic stimulation are high ( 100 - 1,000 ) , and in the most stable preparations with very low recording noise can reach values > > 1,000 ( e.g. , figure 8c ) . notice also that warming expands the cells ' bandwidth of reliable signaling ( snr'bright 1 ) ; for example , the relative difference between the two r1-r6s in figures 8 and 9 , respectively , is 10 hz ( 84 at 20 c and 94 hz at 25 c ) . one can further estimate each cell 's rate of information transfer from its signal - to - noise ratio by using the shannon equation , or by calculating the difference between the responses ' entropy and noise entropy rates through the triple extrapolation method . more details about the information theoretical analyses , and their use and limitations specifically with this method are given in the previous publications . voltage responses of a killer fly r1-r6 photoreceptor and lmc to repeated naturalistic stimulation at 19 c. ( a ) r1-r6 ( gray ) and lmc ( black ) outputs recorded by the same microelectrode from the same fly ; first postsynaptically and later presynaptically , as the electrode was advanced in the eye . ( b ) 20 consecutive pre- ( above ) and postsynaptic ( below ) responses ( light gray traces ) to the same naturalistic stimulus pattern were captured after initial adaptation ( dotted box in a ) . their means are the signals ( the darker traces on top ) , while their respective differences to the individual responses give the noise . ( c ) the corresponding signal - to - noise ratios ( snr ) were calculated as in figures 8c and 9c . lmc output has about a 100 hz broader range of reliable signaling ( snr'lmc1 , up to 234 hz ) than r1-r6 output ( snr'r1 , up to 134 hz ) . both signal - to - noise ratios are high ( snrlmc= 137 , snrr= 627 ) , and as the same microelectrode was used in the recordings , their differences reflect real differences in the pre- and postsynaptic neural outputs . these results imply that the recording system had low noise , and its influence on the analyses was marginal . neuroethological vision studies the method can also be used to record pre- and postsynaptic voltage responses from the compound eyes of different insect species ( figure 10 ) , permitting comparative neuroethological studies of visual information processing . for the presented recording system , the only required adaptation is new preparation - holders , each with a suitably - sized opening for the studied species . they show intracellular voltage responses of a r1-r6 photoreceptor and lmc to identical repetitive light stimulation , as used for the drosophila counterparts in figure 9 , but at 19 c. in this case , both the pre- and postsynaptic data were recorded from the same fly ; one after the other , with the same recording electrode ( filled with 3 m kcl ) first advancing through the lateral lamina before entering the frontal retina . in comparison to the drosophila data at 25 c , the coenosia data - even at the cooler temperature - shows responses with faster dynamics ; expanding the range of reliable signaling ( signal - to - noise ratio > > 1 ) over a broader frequency range . such functional adaptations in neural encoding of naturalistic stimuli are consistent with coenosia 's predatory lifestyle , which require high - precision spatiotemporal information to attain fast aerial hunting behaviors . we have presented the basic key steps of how to use sharp conventional microelectrodes to record intracellular responses of r1-r6 photoreceptors and lmcs in intact fly eyes . this method has been optimized , together with bespoke hardware and software tools , over the last 18 years to provide high - quality long - lasting recordings to answer a wide range of experimental questions . by investing time and resources to construct robust and precise experimental set - ups , and to produce microelectrodes with favorable electrical properties , high - quality recordings can become the norm in any laboratory working on drosophila visual neurophysiology . whilst well - designed recording and light stimulation systems are important for swift execution of different experimental paradigms , there are three procedural steps that are even more critical to achieving successful recordings : ( i ) to make the fly preparation with minimal eye damage , ( ii ) to pull microelectrodes with the right electrical properties , and ( iii ) to drive the recording electrode into the eye without breaking its tip . ultimately , to record meaningful data , the investigator has to understand the physical basis of electrophysiology and how to fabricate suitable microelectrodes for the targeted cell - types . therefore , the limitations of this technique are primarily set by the patience , experience and technical ability of the investigator . because this technique can take a long time to master for small drosophila cells , it is advisable for trainee electrophysiologists to first practice with larger insect eyes , such as the blowfly or locust , using the same rig . once performing high - quality intracellular recordings from the larger photoreceptors and interneurons becomes routine , it is time to move on to the drosophila eye . penetrated drosophila cells can be loaded electrophoretically with dyes , including lucifer yellow or neurobiotin . however , because of the small tip size of the microelectrodes , electrophoresis works less efficiently than with lower resistance electrodes , such as patch - electrodes . furthermore , the dye - filled microelectrodes characteristically have less favorable electrical properties , making it much harder to record high - quality responses with them from drosophila photoreceptors and lmcs . a technical problem that occurs this is often associated with the voltage signal being either constantly drifting or higher / lower than the amplifier 's recording range . on most occasions , this behavior is caused by the recording electrode being blocked ( or its tip being too fine - having too high a resistance or intramural capacitance - to properly conduct fast signal changes ) . although one can try to unblock the tip by buzzing the electrode capacitance , which sometimes works , often the situation is best resolved by simply changing the recording electrode . this may further require parameter adjustments in the microelectrode puller instrument to lower the tip resistance of the new electrodes . the electrode tip can also become blocked in preparations , for which it took too much time to cover the corneal hole by petroleum jelly . prolonged air - contact can dry up the freshly exposed retinal tissue , turning its surface layer into a glue - like substance . if this is the case , the investigator typically sees a red blob of tissue stuck on the recording electrode when pulling it out of the eye . petroleum jelly may provide many benefits for electrophysiological recordings : ( i ) it prevents the coagulation of the hemolymph that could break the electrode tip ; ( ii ) it coats the electrode tip reducing its intramural capacitance , which lowers the electrode 's time constant , and thus has the potential to improve the temporal resolution of the recorded neural signals ; ( iii ) it keeps the electrode tip clean , facilitating penetrations ; and after penetration , ( iv ) it may even help to seal the electrode tip to the cell membrane . the signal can further be unstable or lost when the silver - chloride wire of the electrode - holder is broken or dechloridized ; in which case just replace or rechloridize the old wire . the missing signal can also result from one ( or both ) of the electrode - holders not being securely connected to their jacks . however , it is extremely unusual that a piece of equipment would be malfunctioning . if signal is undetectable and all other possibilities have been exhausted , test that each part of the recording apparatus , including the headstage , amplifier , low - pass filters and ad / da - converters , are connected properly and functioning normally . one way to achieve this is to replace each instrument with another from a rig that is known to operate normally . alternatively , use a signal generator to check the performance of the electronic components one by one . but perhaps the most common technical problem facing the electrophysiologist is that of recording noise . broadly , recording noise is the observed electrical activity other than the direct neuronal response to a given stimulus . because the fly preparation , when properly done , is very stable , the observed noise ( beyond the natural variably of the responses ) most often results from ground - loops in the recording equipment , or is picked up from nearby electrical devices . such noise is typically 50/60 hz mains hum and its harmonics ; but sometimes composed of more complex waveforms . to work out the origin of the noise , remove the fly preparation holder from the set - up , connect the recording and reference electrodes through a drop of fly ringer ( or place them in a small ringer 's solution bath ; see step 1.2.6 ) and record the signal in cc- or bridge - mode . if noise is observable on the recorded signal , this likely means that the noise is external to the fly preparation . another good test for identifying the origin of noise is to replace the electrode - holders with an electric cell model connected to the amplifier . in an ideally configured and grounded set - up , the recorded signal should now be practically noise - free , showing only stochastic bit - noise from the ad - converter ( in the best case not even that ! ) . if noise is still present , then recheck that all rig equipment is properly grounded . a convenient approach to detect ground - loops is to : ( i ) disconnect all the grounding wires from all the parts within the rig ; ( ii ) ensure that , after doing this , every single part is actually isolated from ground , by means of an ohm - meter ; ( iii ) connect the parts , one by one , to the central ground directly , not through any other part of the rig . for example , sometimes moving the computer and monitor further away from the rig can reduce noise ; yet at other times , moving the computer inside the equipment rack reduces noise . it is also worth unplugging nearby equipment to see if noise is reduced , or shield additional components . furthermore , try unplugging or replacing different components of the recording equipment , especially bnc cables ( which can have faulty ground connections ) . if only bit - noise is observed when using the cell model , the initial noise source is either the electrodes or the fly preparation itself . for example , it could be that the reference electrode is inadvertently touching a motor nerve or active muscle fibers inside the head capsule ( or disturbing flight muscles in the thorax - if placed there ) . it is usually simplest to prepare a new fly for recording , taking care to minimize damage to the fly . but if the noise persists and is broadband , it is likely that the electrodes are suboptimal for the experiments ; too sharp / fine ( hence too noisy ) or just wrong for the purpose ; we have even seen quartz - electrodes acting as antennas - picking up faint broadcasting signals ! although iteration of the puller - instrument parameter settings to generate the just right microelectrodes for consistent high - quality recordings from specific cell - types can take a lot of effort , it is worth it . once the recording electrodes are well - tailored for the experiments , they can provide long - lasting recordings of outstanding quality . sharp microelectrode recording techniques can be similarly applied to study neural information processing in multitude of preparations , including different processing layers in the insect eyes and brain . because the microelectrode tips can be made very fine , these typically damage the studied cells less than most patch - clamp applications . importantly , the modern sample - and - hold microelectrode amplifiers enable good control of the tips ' electrical properties . thus , when correctly applied , this technique can provide reliable data from both in vivo or in vitro preparations with high signal - to - noise ratio at sub - millisecond resolution . such precision would be impossible with today 's optical imaging techniques , which are noisier and slower . moreover , the method can be used to characterize small cells ' electrical membrane properties both in current- and voltage - clamp configurations , providing valuable data for biophysical and empirical modeling approaches that link experiments to theory .
voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes . the method described here enables the investigator to measure long - lasting ( from minutes to hours ) high - quality intracellular responses from single drosophila r1-r6 photoreceptors and large monopolar cells ( lmcs ) to light stimuli . because the recording system has low noise , it can be used to study variability among individual cells in the fly eye , and how their outputs reflect the physical properties of the visual environment . we outline all key steps in performing this technique . the basic steps in constructing an appropriate electrophysiology set - up for recording , such as design and selection of the experimental equipment are described . we also explain how to prepare for recording by making appropriate ( sharp ) recording and ( blunt ) reference electrodes . details are given on how to fix an intact fly in a bespoke fly - holder , prepare a small window in its eye and insert a recording electrode through this hole with minimal damage . we explain how to localize the center of a cell 's receptive field , dark- or light - adapt the studied cell , and to record its voltage responses to dynamic light stimuli . finally , we describe the criteria for stable normal recordings , show characteristic high - quality voltage responses of individual cells to different light stimuli , and briefly define how to quantify their signaling performance . many aspects of the method are technically challenging and require practice and patience to master . but once learned and optimized for the investigator 's experimental objectives , it grants outstanding in vivo neurophysiological data .
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Proceed to summarize the following text: gpcrs represent a large family of integral membrane proteins that are encoded by more than 800 genes in the human genome ( zhang et al . , 2006 ) . based on amino acid sequence similarity within the seven - transmembrane ( 7tm ) helical segments , gpcrs are divided into five families : the rhodopsin family ( class a , 701 members ) , the secretin family ( class b , 15 members ) , the adhesion family ( 24 members ) , the glutamate family ( class c , 15 members ) , and the frizzled / taste family ( class f , 24 members ) ( fredriksson et al . , gpcrs recognize a variety of extracellular stimuli , including photons , ions , organic odorants , purines , amines , neurotransmitters , chemokines , hormones , and lipids etc . upon ligand binding , gpcrs undergo conformational changes , coupling to g protein , activating downstream signaling networks and initiating a broad range of physiological and pathological processes ( gether , 2000 ) . gpcrs are important and highly attractive drug targets for numerous human diseases , such as central nervous system disorders , inflammatory diseases , metabolic imbalances , cardiac diseases , monogenic diseases , cancer , and many more ( thomsen et al . , 2005 ) . indeed , 4050 % of the total sales of marketed drugs and 25 % of the top selling drugs target on gpcrs ( schlyer and horuk , 2006 ) gpcr structures are key for not only understanding the molecular mechanisms of gpcr signal transduction , but also developing new therapeutics for the treatment of human diseases . structural studies of gpcr remain challenging and intractable due to the lack of a naturally abundant source of protein , the conformational heterogeneity in solutions , and the relatively small structured polar surface available for forming crystal lattice contacts . the first gpcr structure is the crystal structure of bovine rhodopsin in complex with 11-cis - retinal solved by palczewski and colleagues in 2000 ( palczewski et al . , 2000 ) . after seven - year extensive research and technology development , the high - resolution structure of the human 2-adrenergic receptor ( 2ar ) the first structure of a gpcr bound to a diffusible ligand was obtained ( cherezov et al . , 2007 ) . then , the first active - state gpcr structure of ligand free rhodopsin ( opsin ) was followed in 2008 ( scheerer et al . , 2008 ) . in the past few years , with many technology breakthroughs in membrane protein engineering and crystallography ( caffrey , 2011 ; chae et al . , 2010 ; rosenbaum et al . , 2007 ) , gpcr structure determination has experienced an exponential growth trend . to date , over 100 structures of 28 different class a gpcrs and 5 structures of the transmembrane domain of receptors from classes b , c and f have been determined in complex with ligands of varied pharmacology . these structures shed unprecedented light on structural similarity and diversity of the gpcr super - family , and molecular basis of gpcr ligand recognition , activation , allosteric modulation and dimerization . in this review , we will focus on how the recent structural and biophysical insights contribute to our understanding of gpcr structure - function relationship . we also outline open challenges in gpcr structural studies and discuss new exciting directions for gpcr research to get a comprehensive understanding of biological and therapeutic mechanisms of the gpcr superfamily . so far , a total of 127 gpcr structures ( table 1 ) have been solved for the following class a gpcrs : rhodopsin ( bovine rhodopsin and squid rhodopsin ) ( caffrey , 2011 ; kang et al . , 2015 ; palczewski et al . , 2000 ; park et al . , 2008 ; scheerer et al . , 2008 ; szczepek et al . , 2014 ) ; eight aminergic receptors , including 2-adrenergic receptor ( 2ar ) ( cherezov et al . , 2007 ; rasmussen et al . , 2007 ; 2011 ; rosenbaum et al . , 2007 ; 2011 ) , 1-adrenergic receptor ( 1ar ) ( huang et al . , 2013 ; warne et al . , 2012 ) , h1 histamine receptor ( h1r ) ( shimamura et al . , 2011 ) , d3 dopamine receptor ( d3r ) ( chien et al . , 2010 ) , 5-hydroxytryptamine receptors ( 5-ht1b and 5-ht2b ) ( wacker et al . , 2013 ; wang et al . , 2013a ) and muscarinic acetylcholine receptors ( m2r and m3r ) ( haga et al . , 2012 ; kruse et al . , 2012 ) ; three nucleoside receptors , including a2a adenosine receptor ( a2aar ) ( jaakola et al . , 2008 ; lebon et al . , 2011 ; xu et al . , 2011 ) , p2y1 purinergic receptor ( p2y1r ) ( zhang et al . , 2015a ) and p2y12 purinergic receptor ( p2y12r ) ( zhang et al . , 2014a ; 2014b ) ; ten peptide receptors , including chemokine receptors cxcr4 and ccr5 ( tan et al . , 2013 ; wu et al . , 2010 ) , -opioid receptor ( -or ) ( wu et al . , 2012 ) , -opioid receptor ( -or ) ( manglik et al . , 2012 ) , -opioid receptor ( -or ) ( granier et al . , 2012 ) , nociceptin / orphanin fq peptide receptor ( nop ) ( thompson et al . , 2012 ) , neurotensin receptor 1 ( ntsr1 ) ( white et al . , 2012 ) , angiotensin ii type 1 receptor ( at1r ) ( zhang et al . , 2015b ) , orexin type 2 receptor ( ox2r ) ( yin et al . , 2015 ) , and protease - activated receptor 1 ( par1 ) ( zhang et al . , 2012 ) ; and three lipid - activated gpcrs , including sphingosine-1-phosphate receptor 1 ( s1p1 ) ( hanson et al . , 2012 ) , free fatty acid receptor 1 ( ffar1 ) ( srivastava et al . , 2014 ) and lysophosphatidic acid receptor 1 ( lpa1 ) ( chrencik et al . , 2015 ) . five structures of 7tm domain of the members from classes b , c and f gpcrs , including corticotropin releasing factor receptor 1 ( crf1r ) ( hollenstein et al . , 2013 ) , glucagon receptor ( gcgr ) ( siu et al . , 2013 ) , metabotropic glutamate type 1 receptor ( mglur1 ) ( wu et al . , 2014 ) , metabotropic glutamate type 5 receptor ( mglur5 ) ( dore et al . , 2014 ) , and smoothened receptor ( smo ) ( wang et al . gpcrs share a common structural architecture of 7tm helical bundle connected by three extracellular loops ( ecl1 - 3 ) and three intracellular loops ( icl1 - 3 ) . the n - terminus of gpcr is at the extracellular side , and the c - terminus locates intracellularly . the n - terminus and the extracellular loops are responsible for recognizing a vast variety of ligands and modulating ligand access . the 7tm bundle region forms the structural core , binding ligands and transducing extracellular signals to the intracellular region through conformational changes . the intracellular ( ic ) parts interact with cytosolic g - proteins , arrestins , g protein - coupled receptor kinases ( grks ) and other downstream signaling effectors . one of the most conserved motifs is the d[e]ry [ superscript indicates residue numbering using the ballesteros - weinstein nomenclature ( ballesteros and weinstein , 1995 ) ] motif in helix iii , which often forms a so - called ionic lock by making a salt bridge with d / e in helix vi , for example , in the rhodopsin structure ( palczewski et al . , 2000 ) . the ionic lock was suggested as a characteristic of the inactive conformation of gpcrs , blocking the g - protein binding at the cytoplasmic region ( discussed in the following part ) . the wxp motif in helix vi is also considered as one of the micro - switches that have substantially different conformations in the active state versus the inactive state of the receptor . another conserved motif is the np xxy motif in helix vii , which also plays an important role in gpcr activation ( discussed in the following part ) . in addition to the above conserved motifs in the transmembrane domain , the extracellular loop regions in the gpcr structures also show some similarities . a highly conserved disulfide bond between the residue cys at the extra - cellular tip of helix iii and a cysteine residue in ecl2 was observed in most of the gpcr structures , except for the structure of p2y12r bound to the antagonist azd1283 ( zhang et al . , 2014b ) . this disulfide bond plays an important role in stabilizing the conformation of the extracellular region and shapes the entrance to the ligand - binding pocket . the conformation of the intracellular region of gpcrs is relatively conserved , which may correlate with the limited types of downstream binding partners . in most of the solved gpcr structures , the c - terminus contains a 3 - 4 turn -helix , helix viii , that runs parallel to the membrane and is characterized by a common [ f(rk)xx(fl)xxx ] amphiphilic motif . although all the known gpcr structures are characterized by a similar 7tm topology , remarkable structural diversity was observed , especially in the dynamic extracellular loop region . the largest extracellular loop , ecl2 , structurally differs between receptors , but its conformation is likely to be conserved in a subfamily - specific manner . for example , ecl2 exhibits an -helical structure in adrenergic receptor structures , and a hairpin structure in all the peptide receptor structures . in contrast to ecl2 , ecl1 and ecl3 are relatively short and tend to lack distinct secondary structural elements . besides the structural differences in the extracellular region , striking diversities were also observed in the 7tm helical bundle . the seven -helices display different tilts and rotations in various gpcr structures , which make the ligand - binding pockets of the receptors differ dramatically in size and shape ( discussed in the following part ) . in the known gpcr structures , ligands usually bind to the canonical 7tm helical bundle . however , size , shape and electro - static properties of the ligand - binding pockets are dramatically distinct between different receptors ( fig . , the negative allosteric modulator mavoglurant penetrates into the 7tm bundle of the receptor , locating much deeper compared to the ligands in the other gpcr structures . the ligand - binding sites in the crf1r and mglur1 structures are closer to the extracellular surface than that in mglur5 , but deeper than in the class a gpcrs and smo . in the smo structure , the ligand ly294068 occupies a long and narrow binding pocket , which is connected to the extracellular aqueous environment though a small opening formed by ecl2 and ecl3 ( wang et al . , 2014 ) . remarkable differences were also found in the ligand - binding pockets of class a gpcrs . the ligands of peptide receptors , such as chemokine receptors and opioid receptors , bind to cavities that are shallower and more open compared to the ligand - binding pockets of the receptors with endogenous small - molecule ligands , such as aminergic receptors . this may correlate with the fact that peptides are much larger molecules that need more binding space than the small molecules and can not enter deeply into the 7tm bundle like the small - molecule ligands . despite of the high overall structural similarity , ligand - binding modes can be distinct between receptors in the same sub - family ( fig . 2 ) . the structures of purinergic receptors p2y1r and p2y12r reveal very different features in binding their nucleotide - like ligands , although these two receptors recognize the same endogenous ligand adp . the ligand - binding sites for the nucleotide - like antagonist mrs2500 in p2y1r and the nucleotide agonist 2mesadp in p2y12r are spatially distinct with only a minor overlap . in the p2y1r structure , the adenine group of mrs2500 is closed to helices vi and vii , whereas the adenine ring of 2mesadp reaches deep into the binding pocket and interacts with helices iii and iv in the p2y12r structure ( zhang et al . the antagonist it1 t in the cxcr4 structure occupies part of the binding pocket formed by helices i , ii , iii and vii and ecl2 , while in the ccr5 structure , the ligand maraviroc locates in a deeper binding site and occupies a larger area of the pocket defined by residues from helices i , ii , iii , v , vi and vii , making no contacts with the extracellular loops ( tan et al . , 2013 additionally , there are differences on the approach routes that gpcr ligands enter the binding pockets . in most of the gpcr structures , there is a large extracellular vestibule as part of an extended hydrophilic channel to the orthosteric binding pocket where the ligands gain access into the binding pockets . however , in the lipid - activated gpcrs , such as s1p1 and ffar1 , receptor s n - terminus or extracellular loops form a cap - like structure over the ligand - binding pocket , blocking the entrance to the ligand - binding cavity through the extracellular region of the receptor ( hanson et al . , 2012 ; srivastava et al . , the ligands of these receptors are highly lipophilic and bind to the receptors mainly through hydrophobic interactions . this ligand - binding mode and the hydrophobic nature of the ligands suggest that these ligands most likely gain access into the binding pockets through the lipid bilayer . a similar approach route of ligand was proposed in the structure of p2y1r bound to the non - nucleotide antagonist bptu . in this structure , bptu binds to a shallow and highly hydrophobic pocket on the external receptor interface with the lipid bilayer . this is the first structurally characterized selective gpcr ligand located entirely outside of the 7tm helical bundle ( zhang et al . , 2015a ) . it is widely believed that there is an equilibrium between two conformations of gpcr , the inactive state ( r ) and the active state ( r ) . in the absence of ligands , the efficacy of ligands reflects their ability to alter the equilibrium between the inactive state and the active state . full agonists preferentially bind to and stabilize the active conformation , and shift the equilibrium to r , while inverse agonists stabilize the inactive conformation , and shift the equilibrium to r. partial agonists have some affinity for both r and r , and are therefore less effective in shifting the equilibrium towards r ( kobilka and deupi , 2007 ) . to date , the structures of several different gpcrs , rhodopsin , 1ar , 2ar , a2aar , m2r , p2y12r and -or , have been determined in both antagonist - bound and agonist - bound states ( haga et al . , 2012 ; , 2008 ; kang et al . , 2015 ; lebon et al . , 2011 ; 2012 ; manglik et al . , 2012 ; palczewski et al . , 2000 ; rosenbaum et al . , 2007 ; xu et al . , 2011 ; zhang et al these structures provide insights into the molecular mechanisms about how the ligands bind at the extracellular side of the receptors and trigger conformational changes in the receptors 7tm domains , where g - protein and other effectors bind and initiate a series of downstream signaling pathways . in spite of the remarkable diversity of ligands and ligand - binding sites of gpcrs , the receptors share similar activation mechanisms . some conserved motifs in gpcrs are defined as molecular micro - switches , which are believed to be important in the regulation of receptor activation and signaling , acting as switches between inactive states and active states ( nygaard et al . , 2009 ) . in the rhodopsin and a2aar structures , r in the d[e]r y motif interacts with e in helix vi to form the ionic lock , which is broken in the active structures ( scheerer et al . , 2008 ; as seen in the rhodopsin and a2aar structures , a conformational change of the side chain of w is induced by interacting with agonists , leading to a movement of helix vi , which is considered as a major ligand - dependent trigger in gpcr activation ( park et al . , 2008 ; xu et al . , 2011 ) additionally , the npxxy motif was also proposed as one of the important micro - switches . in the structure of ligand - free opsin , the y residue rotates to face into the helical bundle , blocking helix vi from moving back towards helix iii to adopt the inactive conformation ( park et al . , 2008 ) . multiple biochemical and biophysical studies indicate that receptor activation is associated with a relatively large - scale rearrangement of the transmembrane helices , leading to different receptor conformations and differential effects on downstream signaling proteins ( kobilka , 2007 ; nygaard et al . , 2009 ) . such conformational changes have been observed in the structures of several different gpcrs , such as rhodopsin , a2aar , 2ar and 1ar ( huang et al . , 2013 ; the helical rearrangements include an outward movement of helices v and vi and an inward motion of helix vii , leading to an opening of the helical bundle at the cytoplasmic side , which facilitates the g - protein coupling and subsequent activation ( fig . however , in the p2y12r structures , remarkable conformational changes occur in the extracellular part . compared with the antagonist - bound p2y12r structure , the extracellular tips of helices vi and vii in the agonist - bound structure shift 510 towards the central axis of the 7tm helical bundle ( fig . this finding suggests that not only the intracellular parts but also the extracellular parts of gpcrs play critical roles in receptor activation . upon activation , gpcrs couple to g protein , changing gdp that binds to g protein to gtp . the active gpcrs can also interact with grks , which phosphorylate gpcrs to trigger a high affinity binding to arrestins . this blocks the interaction between the receptors and g protein , turning off g - protein signaling , and leads to gpcr desensitization ( nygaard et al . , the complex structures of 2ar bound to gs protein ( 2ar - gs ) ( rasmussen et al . , 2011 ) , bovine rhodopsin in complex with a g - protein peptide ( rhodopsin - gct ) ( scheerer et al . , 2008 ) and human rhodopsin bound to a pre - activated mouse visual arrestin ( kang et al . , 2015 ) provide molecular basis of gpcr signaling . in the structures of 2ar - gs and rhodopsin - gct , the c - terminus of g inserts into the intracellular side of the receptor s 7tm helical bundle , leading to the rearrangement of helices v , vi and vii . the rhodopsin - arrestin structure shows a large rhodopsin - arrestin interface formed by icl1 , n - terminus of helix viii and the c - terminal region of helix vii . these structures provide insights into the interactions between gpcrs and g - proteins , as well as arrestins . however , more complex structures between different gpcrs and various g proteins , arrestins , as well as grks are desperately needed for better understanding the molecular mechanisms of gpcr signaling transduction . gpcrs can not only recognize orthosteric ligands but also are affected by a variety of endogenous allosteric modulators , including lipids , ions , waters and sterols ( chini and parenti , 2009 ) . these modulators regulate receptor function by binding to alternative regions in the receptors , allosteric binding sites , instead of the conventional orthosteric binding sites . in recent years , there has been increasing therapeutic interest in ligands that bind to the allosteric sites of gpcrs . allosteric ligands can potentially be more selective than the orthosteric ligands because of their little conserved inherent activity , making them highly beneficial therapeutic candidates with less adverse effects from overdose . allosteric ligands have been identified for many gpcrs , such as adenosine receptors , histamine receptors , dopamine receptors , serotonin receptors , muscarinic receptors , chemokine receptors and class c gpcrs . the previously solved gpcr structures reveal various allosteric binding sites within the 7tm helical bundle ( in ccr5 , mglur1 and mglur5 ) ( dore et al . , 2014 ; tan et al . , 2013 ; wu et al . , 2014 ) , in the extracellular region of the receptor ( in m2r ) ( haga et al . , 2012 ) , or on the external lipidic interface of the transmembrane domain ( in p2y1r ) ( zhang et al . the gpcr structures deepen our understanding about gpcr allosteric modulation , and open the door to high selective and novel therapeutic agents for the treatment of multiple human disorders . the a2aar structure suggests that sodium ion may act as an allosteric modulator to alter the dynamics and activation profiles of gpcrs ( gutierrez - de - teran et al . , 2013 ) . in the 2ar structure , the specific cholesterol binding modulates receptor thermostability and binding affinity for inverse agonists ( cherezov et al . , 2007 ) the ccr5 structure demonstrates that the allosteric inverse agonist maraviroc inhibits receptor function by stabilizing the receptor in an inactive conformation ( tan et al . , 2013 ) . the unique allosteric binding site of the p2y1r antagonist bptu on the external receptor surface broaden the new scope of future gpcr ligand discovery outside of the canonical orthosteric ligand - binding pocket ( zhang et al . a growing body of biochemical and biophysical evidence indicates that many gpcrs can form homo- or hetero - dimers or larger oligomers ( lee et al . , 2003 ) . these dimers and oligomers have been proposed to be important for modulation of gpcr signal transduction and mediation of crosstalk between gpcr pathways ( lohse , 2010 ; milligan , 2009 ; pin et al . , 2004 ) . parallel dimers with substantial receptor - receptor interfaces have been observed in the structures of some gpcrs , including 2ar , cxcr4 , -or , -or and mglur1 ( manglik et al . , 2012 ; rasmussen et al . , 2007 ; wu et al . , 2010 ; 2012 ; 2014 ) . in the rhodopsin and -or structures , a parallel receptor dimer is formed via helices i , ii and viii . in the cxcr4 and -or structures , a parallel , symmetric dimer is formed via helices v and vi . a receptor dimer mediated by interactions between receptor s helix i and cholesterol molecules was observed in the 7tm domain structure of mglur1 . in the 2ar structure , the dimer is mediated by ordered lipids consisting of two cholesterol molecules and two palmitic acid molecules in the c - terminal region of the receptor . although the above structural information provides insights into the gpcr dimerization , whether the dimerization is necessary for signal transduction and how do the dimers function in physiological state are still unclear . more structural evidence of gpcr dimerization is needed to elucidate what is likely to be a common underlying mechanism for gpcr dimerization . recent tremendous breakthroughs in gpcr structural studies provide us opportunities to learn the structural features of gpcrs and the molecular mechanisms of various gpcr physiological behaviors , such as ligand recognition , receptor activation , allosteric modulation and dimerization , etc . however , the current structural information is far away from enough to understand all the biological functions of gpcrs . more structures from different receptor sub - families , such as adhesion receptors , olfactory receptors and many orphan receptors , are essential to fully understand the ligand - binding landscape of the gpcr superfamily . although several trans - membrane domain structures of classes b , c and f receptors have been solved , the full - length structures of non - rhodopsin family gpcrs are necessitous to reveal the mechanisms of receptor activation for these specific families . determining the complex structures of gpcrs coupling to the intracellular signaling effectors is still considered as one of the major tasks of the gpcr structural studies , but remains extremely challenging . additionally , the structures of gpcr homo- or heterodimers are key for understanding how gpcrs regulate the cell signaling by changing their stoichiometry . will immensely facilitate structure - based drug discovery and certainly open up further therapeutic opportunities for many severe diseases .
g protein - coupled receptors ( gpcrs ) constitute the largest and the most physiologically important membrane protein family that recognizes a variety of environmental stimuli , and are drug targets in the treatment of numerous diseases . recent progress on gpcr structural studies shed light on molecular mechanisms of gpcr ligand recognition , activation and allosteric modulation , as well as structural basis of gpcr dimerization . in this review , we will discuss the structural features of gpcrs and structural insights of different aspects of gpcr biological functions .
You are an expert at summarizing long articles. Proceed to summarize the following text: in acute coronary syndromes , unstable atherothrombotic plaques contain more lipids and thrombi and less calcification . . iatrogenic rupture of atherosclerotic plaques during percutaneous coronary intervention can also result in coronary microembolization ( cme ) , in spite of successful recanalization , leading to inadequate myocardial perfusion , continuous myocardial ischemia , progressive contractile dysfunction , and fatal arrhythmia . cme drew the attention of researchers about two decades ago with the awareness of its clinical frequency and importance . experimental animal models are required to understand the pathophysiological , morphological , molecular , and biological changes and to test the efficacy of new therapeutic interventions . embolization by intracoronary infusion of microspheres into the coronary microcirculation has been used in large laboratory animals , mostly dogs and swine . a mouse model of cme was established in the present study by an intraventricular injection of microspheres during occlusion of the ascending aorta . based on the average diameter and dosage used in cme , a total of 30 c57bl/6 male mice weighing 2025 g were provided by the department of laboratory animal science , fudan university , shanghai , china , and kept under controlled conditions for temperature , humidity , and light , with standard chow and water available ad libitum . the mice were randomly divided into five groups : 9 m/500,000 , 9 m/800,000 , 17 m/200,000 , 17 m/500,000 , and sham groups . the study accorded with the guide for the care and use of laboratory animals published by the us national institutes of health ( nih publication no . 85 - 23 , revised 1996 ) , and it was approved by the animal care and use committee of fudan university , china . the original liquid of white - stained polystyrene microspheres ( dynospheres ; dyno particles , lillestrm , norway ) was diluted with the normal saline solution . microspheres of both sizes were counted using a hemocytometer under an optical microscope to acquire different concentrations of microsphere diluents , including 500,000/50 l and 800,000/50 l for microspheres at an average diameter of 9 m and 200,000/50 l and 500,000/50 l for microspheres at an average diameter of 17 m . , il , usa ) in oxygen , intubated and ventilated with a small animal ventilator ( type 7025 , ugo basile , comerio , italy ) at 100 breaths per minute . anesthesia of the mice was maintained with the inhalation of 1.5% isoflurane during the procedures . the heart and the ascending aorta were exposed through a middle line thoracotomy and then a sternotomy at the second and third intercostal spaces . a silk suture ( size # 5 ) was placed afterward under the aortic root for occluding the ascending aorta [ figure 1a ] . subsequently , diluted white - stained polystyrene microspheres at a volume of 50 l with different diameters and dosages were injected as a bolus with a 29-gauge needle into the left ventricular chamber during 15-s occlusion of the ascending aorta [ figure 1b ] . ( a ) a # 5 silk suture has been placed under the aortic root ( solid arrow ) after thoracotomy , and 500,000 white - stained polystyrene microspheres with an average diameter of 9 m at a volume of 50 l have been prepared ( dotted arrow ) . ( b ) the injection of microspheres into the left ventricular chamber from the apex while the ascending aorta was occluded . the general conditions and mobility , as well as the number of the surviving and dead mice , were observed and recorded every day . the surviving mice were sacrificed by cervical dislocation 3 and 10 days after the surgery . for all the cases of unexpected death , thoracotomy was performed to see whether operation - related complications such as massive bleeding or pulmonary artery or aorta injury existed . the heart tissues of the mice were acquired and sent for histological analyses as soon as possible . most heart tissues were fixed with 10% formaldehyde , embedded in paraffin , and then sectioned into slices of 5 m thickness for hematoxylin and eosin ( he ) staining and masson trichrome staining . an average of three slices from different parts of the heart of every mouse were analyzed for the extent of inflammatory cellular infiltration and fibrin deposition using the imagepro plus 6.0 software ( media cybernetics , inc . the extent of inflammatory cellular infiltration was expressed as a percentage calculated by dividing the area of inflammatory cellular infiltration by the total area of heart tissue in the slice ; the same algorithm was used for the degree of fibrin deposition . a small part of heart tissues from every mouse was processed and fixed with 2.5% glutaraldehyde and then delivered to the electron microscope room , shanghai medical college , fudan university , for scanning transmission electron microscope detections to observe ultrastructural changes . the brain tissues of the paralyzed mice after operation were also collected for he staining . moreover , the kidney tissues of the mice from cme groups were harvested for he staining to understand the distribution of the microspheres in the systemic circulation . groups were compared by one - way analysis of variance , and bonferroni 's test was performed to identify differences between groups . all p values were two - sided , and p < 0.05 was considered statistically significant . based on the average diameter and dosage used in cme , a total of 30 c57bl/6 male mice weighing 2025 g were provided by the department of laboratory animal science , fudan university , shanghai , china , and kept under controlled conditions for temperature , humidity , and light , with standard chow and water available ad libitum . the mice were randomly divided into five groups : 9 m/500,000 , 9 m/800,000 , 17 m/200,000 , 17 m/500,000 , and sham groups . the study accorded with the guide for the care and use of laboratory animals published by the us national institutes of health ( nih publication no . 85 - 23 , revised 1996 ) , and it was approved by the animal care and use committee of fudan university , china . the original liquid of white - stained polystyrene microspheres ( dynospheres ; dyno particles , lillestrm , norway ) was diluted with the normal saline solution . microspheres of both sizes were counted using a hemocytometer under an optical microscope to acquire different concentrations of microsphere diluents , including 500,000/50 l and 800,000/50 l for microspheres at an average diameter of 9 m and 200,000/50 l and 500,000/50 l for microspheres at an average diameter of 17 m . , il , usa ) in oxygen , intubated and ventilated with a small animal ventilator ( type 7025 , ugo basile , comerio , italy ) at 100 breaths per minute . anesthesia of the mice was maintained with the inhalation of 1.5% isoflurane during the procedures . the heart and the ascending aorta were exposed through a middle line thoracotomy and then a sternotomy at the second and third intercostal spaces . a silk suture ( size # 5 ) was placed afterward under the aortic root for occluding the ascending aorta [ figure 1a ] . subsequently , diluted white - stained polystyrene microspheres at a volume of 50 l with different diameters and dosages were injected as a bolus with a 29-gauge needle into the left ventricular chamber during 15-s occlusion of the ascending aorta [ figure 1b ] . ( a ) a # 5 silk suture has been placed under the aortic root ( solid arrow ) after thoracotomy , and 500,000 white - stained polystyrene microspheres with an average diameter of 9 m at a volume of 50 l have been prepared ( dotted arrow ) . ( b ) the injection of microspheres into the left ventricular chamber from the apex while the ascending aorta was occluded . the general conditions and mobility , as well as the number of the surviving and dead mice , were observed and recorded every day . the surviving mice were sacrificed by cervical dislocation 3 and 10 days after the surgery . for all the cases of unexpected death , thoracotomy was performed to see whether operation - related complications such as massive bleeding or pulmonary artery or aorta injury existed . the heart tissues of the mice were acquired and sent for histological analyses as soon as possible . most heart tissues were fixed with 10% formaldehyde , embedded in paraffin , and then sectioned into slices of 5 m thickness for hematoxylin and eosin ( he ) staining and masson trichrome staining . an average of three slices from different parts of the heart of every mouse were analyzed for the extent of inflammatory cellular infiltration and fibrin deposition using the imagepro plus 6.0 software ( media cybernetics , inc . the extent of inflammatory cellular infiltration was expressed as a percentage calculated by dividing the area of inflammatory cellular infiltration by the total area of heart tissue in the slice ; the same algorithm was used for the degree of fibrin deposition . a small part of heart tissues from every mouse was processed and fixed with 2.5% glutaraldehyde and then delivered to the electron microscope room , shanghai medical college , fudan university , for scanning transmission electron microscope detections to observe ultrastructural changes . the brain tissues of the paralyzed mice after operation were also collected for he staining . moreover , the kidney tissues of the mice from cme groups were harvested for he staining to understand the distribution of the microspheres in the systemic circulation . groups were compared by one - way analysis of variance , and bonferroni 's test was performed to identify differences between groups . all p values were two - sided , and p < 0.05 was considered statistically significant . one mouse ( 1/6 ) in the 9 m/800,000 group and one ( 1/6 ) in the 17 m/500,000 group had poor performance after cme . no massive bleeding in the chest or pulmonary artery or aorta injuries was observed . the others ( 5/6 ) in the 9 m/800,000 group survived . two mice ( 2/6 ) in the 17 m/500,000 group and one ( 1/6 ) in the 17 m/200,000 group were paralyzed , presenting with the abnormality in the right hind leg movement and inability to feed themselves ; they died the next day . moreover , three mice ( 3/6 ) in the 17 m/500,000 group and one ( 1/6 ) in the 17 m/200,000 group died immediately due to cardiac arrest after injecting the microspheres . the others ( 4/6 ) in the 17 m/200,000 group survived . in consequence of the unexpected deaths , three , two , one , and three mice were finally sacrificed 3 days after the surgery in the 9 m/500,000 , 9 m/800,000 , 17 m/200,000 , and sham groups , respectively ; three mice in each of these groups were sacrificed 10 days after the surgery . survival analysis demonstrated that the cumulative survival rate of the 17 m/500,000 group was significantly lower than that of the sham group ( 0/6 vs. 6/6 , p = 0.001 ) [ figure 2 ] . the cumulative survival rate of the 17 m/200,000 group was lower than those of the sham and 9 m groups ( cumulative survival rate of the 17 m/200,000 , 9 m/800,000 , 9 m/500,000 , and sham groups was 4/6 , 5/6 , 6/6 , and 6/6 , respectively ) , but the statistical difference was not significant ( 17 m/200,000 vs. sham , p = 0.139 ) . survival analysis of coronary embolization with different sizes and quantities ( n = 6 in each group ) . * compared to the sham group , the cumulative survival rate was significantly lower in the 17 m/500,000 group ( p = 0.001 ) . there were no significant differences among the 17 m/200,000 , 9 m/800,000 , 9 m/500,000 , and sham groups . he staining showed numerous microspheres in the coronary microcirculation without obvious inflammatory cell infiltration in the 17 m/500,000 group on both the day and the next day of cme . multiple myocardial necroses of varying sizes with inflammatory cell infiltration and collagen deposition were revealed in all the cme groups 3 and 10 days after the surgery . these changes were not observed in the sham group [ figures 4 - 7 ] . histopathological examinations showed significant inflammatory infiltration and collagen deposition 3 and 10 days after the surgery in the 17 m/200,000 ( inflammatory infiltration : 15.16% 7.73% at day 10 ; collagen deposition : 16.27% 5.74% at day 10 ) , 9 m/800,000 ( inflammatory infiltration : 9.11% 4.96% at day 10 ; collagen deposition : 9.19% 4.52% at day 10 ) , and 9 m/500,000 groups ( inflammatory infiltration : 10.43% 5.86% at day 10 ; collagen deposition : 9.15% 4.81% at day 10 ) compared with the sham group ( inflammatory infiltration : 1.24% 0.23% at day 10 ; collagen deposition : 1.21% 0.17% at day 10 ) . the extent of inflammatory cellular infiltration and collagen deposition in the 17 m/200,000 group was more severe than that in the 9 m/800,000 and 9 m/500,000 groups , both 3 and 10 days after the surgery . no significant differences in histopathological variations were observed between the 9 m/800,000 and 9 m/500,000 groups [ figures 8 and 9 ] . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in 17 m/500,000 group 1 day after coronary microembolization operation . ( a - c ) images in he staining , with original magnification 25 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining , with original magnification 25 , 200 , and 400 , respectively . numerous microspheres filled in the coronary microcirculation without obvious inflammatory cell infiltration or collagen deposition . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in 17 m/200,000 group 1 , 3 , and 10 days after the surgery . ( a - c ) images in he staining 1 day after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining 1 day after surgery , with original magnification 25 , 200 , and 400 , respectively . ( g - l ) 3 days after the surgery and ( m - r ) 10 days after the surgery . the images showed myocardial necroses of varying sizes with inflammatory cell infiltration and collagen deposition 3 and 10 days after the surgery in 17 m/200,000 group . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in the 9 m/800,000 group 1 , 3 , and 10 days after the surgery . ( a - c ) images in he staining 1 day after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining 1 day after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( g - l ) 3 days after the surgery and ( m - r ) 10 days after the surgery . the images showed myocardial necroses of varying sizes with inflammatory cell infiltration and collagen deposition 3 and 10 days after the surgery in the 9 m/800,000 group . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in the 9 m/500,000 group 3 and 10 days after the surgery . ( a - c ) images in he staining 3 days after the surgery , with original magnification 5 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining 3 days after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( the images showed myocardial necroses of varying sizes with inflammatory cell infiltration and collagen deposition 3 and 10 days after the surgery in the 9 m/500,000 group . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in the sham group 3 and 10 days after the surgery . ( a - c ) images in he staining 3 days after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining 3 days after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( g - l ) 10 days after the surgery . the other three groups had significant inflammatory infiltration 3 and 10 days after the surgery ( * p < 0.05 ) ; there was no significant difference between the 9 m/800,000 and 9 m/500,000 groups ; the area of inflammatory cellular infiltration was more in the 17 m/200,000 group than 9 m/800,000 and 9 m 500,000 groups 3 and 10 days after the surgery ( p < 0.05 ) . comparison of the extent of fibrin deposition among groups . compared to the sham group , the other three groups had significant fibrin deposition 3 and 10 days after the surgery ( * p < 0.05 ) ; there was no significant difference between the 9 m/800,000 and 9 m/500,000 groups ; the extent of fibrin deposition was more severe in the 17 m/200,000 group than 9 m/800,000 and 9 m 500,000 groups 3 and 10 days after the surgery ( p < 0.05 ) . a few singular microspheres were found in the kidney specimens of both the 9 m and 17 m groups [ figure 10 ] . he staining of the brain tissues of the paralyzed mice revealed numerous microspheres at an average diameter of 17 m accompanied by a disorganized structure of brain tissues and focal vacuole - like changes [ figure 11 ] . a few singular microspheres ( arrow ) were found in these kidney specimens , indicating part of the microspheres went into the systemic circulation with blood flow . the representative images of brain tissue of the paralyzed mice in the 17 m/200,000 group with he staining ( original magnification 200 ) . ( a - c ) plentiful microspheres at an average diameter of 17 m ( arrow ) accompanied by disorganized structure of brain tissues , and focal vacuole - like changes were observed . the neatly arranged myoneme and densely distributed mitochondria were observed in the normal myocardium [ figure 12a and 12b ] . polystyrene microspheres were packed in capillaries [ figure 12c ] , accompanied by platelet adhesion [ figure 12 g ] , aggregation [ figure 12h ] , and microthrombi formation [ figure 12 g and 12h ] in the coronary embolization group . mitochondrial swelling [ figure 12d ] , release of organelles , disorganized myocardial fibers , widened intercellular space [ figure 12e and 12f ] , and inflammatory cell infiltration [ figure 12h ] were detected . ( a and b ) normal ultrastructure in the sham group ; ( c ) the arrow indicates polystyrene microspheres packed in capillaries ; ( d ) the arrow indicates swelling mitochondria ; ( e ) the arrow indicates destroyed myocardiocyte and organelles release ; ( f ) the arrow indicates the ruptured myocardial cell membrane ; ( g ) the arrow indicates platelet adhesion and microemboli formation ; ( h ) the arrow indicates platelet aggregation and leukocytes outside of the vessel . the scale bar in ( a ) indicates 10 m ; one mouse ( 1/6 ) in the 9 m/800,000 group and one ( 1/6 ) in the 17 m/500,000 group had poor performance after cme . no massive bleeding in the chest or pulmonary artery or aorta injuries was observed . the others ( 5/6 ) in the 9 m/800,000 group survived . two mice ( 2/6 ) in the 17 m/500,000 group and one ( 1/6 ) in the 17 m/200,000 group were paralyzed , presenting with the abnormality in the right hind leg movement and inability to feed themselves ; they died the next day . moreover , three mice ( 3/6 ) in the 17 m/500,000 group and one ( 1/6 ) in the 17 m/200,000 group died immediately due to cardiac arrest after injecting the microspheres . the others ( 4/6 ) in the 17 m/200,000 group survived . in consequence of the unexpected deaths , three , two , one , and three mice were finally sacrificed 3 days after the surgery in the 9 m/500,000 , 9 m/800,000 , 17 m/200,000 , and sham groups , respectively ; three mice in each of these groups were sacrificed 10 days after the surgery . survival analysis demonstrated that the cumulative survival rate of the 17 m/500,000 group was significantly lower than that of the sham group ( 0/6 vs. 6/6 , p = 0.001 ) [ figure 2 ] . the cumulative survival rate of the 17 m/200,000 group was lower than those of the sham and 9 m groups ( cumulative survival rate of the 17 m/200,000 , 9 m/800,000 , 9 m/500,000 , and sham groups was 4/6 , 5/6 , 6/6 , and 6/6 , respectively ) , but the statistical difference was not significant ( 17 m/200,000 vs. sham , p = 0.139 ) . survival analysis of coronary embolization with different sizes and quantities ( n = 6 in each group ) . * compared to the sham group , the cumulative survival rate was significantly lower in the 17 m/500,000 group ( p = 0.001 ) . there were no significant differences among the 17 m/200,000 , 9 m/800,000 , 9 m/500,000 , and sham groups . he staining showed numerous microspheres in the coronary microcirculation without obvious inflammatory cell infiltration in the 17 m/500,000 group on both the day and the next day of cme . multiple myocardial necroses of varying sizes with inflammatory cell infiltration and collagen deposition were revealed in all the cme groups 3 and 10 days after the surgery . these changes were not observed in the sham group [ figures 4 - 7 ] . histopathological examinations showed significant inflammatory infiltration and collagen deposition 3 and 10 days after the surgery in the 17 m/200,000 ( inflammatory infiltration : 15.16% 7.73% at day 10 ; collagen deposition : 16.27% 5.74% at day 10 ) , 9 m/800,000 ( inflammatory infiltration : 9.11% 4.96% at day 10 ; collagen deposition : 9.19% 4.52% at day 10 ) , and 9 m/500,000 groups ( inflammatory infiltration : 10.43% 5.86% at day 10 ; collagen deposition : 9.15% 4.81% at day 10 ) compared with the sham group ( inflammatory infiltration : 1.24% 0.23% at day 10 ; collagen deposition : 1.21% 0.17% at day 10 ) . the extent of inflammatory cellular infiltration and collagen deposition in the 17 m/200,000 group was more severe than that in the 9 m/800,000 and 9 m/500,000 groups , both 3 and 10 days after the surgery . no significant differences in histopathological variations were observed between the 9 m/800,000 and 9 m/500,000 groups [ figures 8 and 9 ] . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in 17 m/500,000 group 1 day after coronary microembolization operation . ( a - c ) images in he staining , with original magnification 25 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining , with original magnification 25 , 200 , and 400 , respectively . numerous microspheres filled in the coronary microcirculation without obvious inflammatory cell infiltration or collagen deposition . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in 17 m/200,000 group 1 , 3 , and 10 days after the surgery . ( a - c ) images in he staining 1 day after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining 1 day after surgery , with original magnification 25 , 200 , and 400 , respectively . ( g - l ) 3 days after the surgery and ( m - r ) 10 days after the surgery . the images showed myocardial necroses of varying sizes with inflammatory cell infiltration and collagen deposition 3 and 10 days after the surgery in 17 m/200,000 group . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in the 9 m/800,000 group 1 , 3 , and 10 days after the surgery . ( a - c ) images in he staining 1 day after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining 1 day after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( g - l ) 3 days after the surgery and ( m - r ) 10 days after the surgery . the images showed myocardial necroses of varying sizes with inflammatory cell infiltration and collagen deposition 3 and 10 days after the surgery in the 9 m/800,000 group . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in the 9 m/500,000 group 3 and 10 days after the surgery . ( a - c ) images in he staining 3 days after the surgery , with original magnification 5 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining 3 days after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( g - l ) 10 days after the surgery . the images showed myocardial necroses of varying sizes with inflammatory cell infiltration and collagen deposition 3 and 10 days after the surgery in the 9 m/500,000 group . the representative images of myocardium of the left ventricle with he staining or masson trichrome staining in the sham group 3 and 10 days after the surgery . ( a - c ) images in he staining 3 days after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( d - f ) images in masson trichrome staining 3 days after the surgery , with original magnification 25 , 200 , and 400 , respectively ; ( g - l ) 10 days after the surgery . the other three groups had significant inflammatory infiltration 3 and 10 days after the surgery ( * p < 0.05 ) ; there was no significant difference between the 9 m/800,000 and 9 m/500,000 groups ; the area of inflammatory cellular infiltration was more in the 17 m/200,000 group than 9 m/800,000 and 9 m 500,000 groups 3 and 10 days after the surgery ( p < 0.05 ) . comparison of the extent of fibrin deposition among groups . compared to the sham group , the other three groups had significant fibrin deposition 3 and 10 days after the surgery ( * p < 0.05 ) ; there was no significant difference between the 9 m/800,000 and 9 m/500,000 groups ; the extent of fibrin deposition was more severe in the 17 m/200,000 group than 9 m/800,000 and 9 m 500,000 groups 3 and 10 days after the surgery ( p < 0.05 ) . a few singular microspheres were found in the kidney specimens of both the 9 m and 17 m groups [ figure 10 ] . he staining of the brain tissues of the paralyzed mice revealed numerous microspheres at an average diameter of 17 m accompanied by a disorganized structure of brain tissues and focal vacuole - like changes [ figure 11 ] . . a few singular microspheres ( arrow ) were found in these kidney specimens , indicating part of the microspheres went into the systemic circulation with blood flow . the representative images of brain tissue of the paralyzed mice in the 17 m/200,000 group with he staining ( original magnification 200 ) . ( a - c ) plentiful microspheres at an average diameter of 17 m ( arrow ) accompanied by disorganized structure of brain tissues , and focal vacuole - like changes were observed . the neatly arranged myoneme and densely distributed mitochondria were observed in the normal myocardium [ figure 12a and 12b ] . polystyrene microspheres were packed in capillaries [ figure 12c ] , accompanied by platelet adhesion [ figure 12 g ] , aggregation [ figure 12h ] , and microthrombi formation [ figure 12 g and 12h ] in the coronary embolization group . mitochondrial swelling [ figure 12d ] , release of organelles , disorganized myocardial fibers , widened intercellular space [ figure 12e and 12f ] , and inflammatory cell infiltration [ figure 12h ] were detected . ( a and b ) normal ultrastructure in the sham group ; ( c ) the arrow indicates polystyrene microspheres packed in capillaries ; ( d ) the arrow indicates swelling mitochondria ; ( e ) the arrow indicates destroyed myocardiocyte and organelles release ; ( f ) the arrow indicates the ruptured myocardial cell membrane ; ( g ) the arrow indicates platelet adhesion and microemboli formation ; ( h ) the arrow indicates platelet aggregation and leukocytes outside of the vessel . the scale bar in ( a ) indicates 10 m ; the scale bars in ( b - h ) indicate 5 m . to the best of our knowledge , mice have seldom been used to construct the model of cme so far . the present study demonstrated that the mouse model of cme could be established by the unselective intracoronary fusion of polystyrene microspheres with the occluded ascending aorta through injection from the apex . the operation could be successfully done with a high survival rate in the 9 m/500,000 group . based on the general conditions , postoperative survival rates , and histopathological changes , 500,000 polystyrene microspheres at an average diameter of 9 m lycopodium spores were the first materials to be used to embolize coronary arteries to induce myocardial ischemia . then , different sizes of microspheres replaced the lycopodium spores and were widely applied in the experimental coronary embolization . nevertheless , lycopodium spores or microspheres did not resemble microemboli found in the hearts of patients who died of sudden death or acute coronary syndrome at autopsy . the microemboli , composed of platelets , fibrin , hemocytes , and atherosclerotic plaque materials , including cholesterol crystals , induced a marked inflammatory response characterized by leukocyte infiltration . it is hard to duplicate the pathophysiological process of cme because the microemboli identified in the patients who suffered from sudden death or acute coronary syndrome can not be reproduced . however , the use of microspheres in animals induces similar microinfarcts as reported in the patients hearts , which was confirmed in the present mouse model of cme . platelet adhesion , aggregation , and microthrombi formation , accompanied by the myocardial cell injury , were detected by scanning transmission electron microscopy . moreover , the area of the microinfarcts induced by 9 m/500,000 microspheres in the present study was similar to that in the widely used pig models . the homologous microthrombotic particles were prepared based on the venous blood clotting , followed by screening out of the particles with appropriate sizes through a filter therefore , it has been used in constructing rat models of coronary artery microthrombosis , cerebral infarction , and peripheral arterial occlusive disease . injection of sodium laurate can cause more severe myocardial infarct and inflammation compared with microspheres . moreover , sodium laurate goes into systemic circulation and destroys the integrity of vascular walls , followed by serious and progressive in situ thrombosis and organ dysfunction . hence , it seems that an ideal animal model of cme does not exist at present . a marked difference exists between big and small animals in the method of injecting microspheres . for big animals , such as dogs , swine , mini swine , calves , and sheep , a selective injection of microspheres into the coronary artery , usually the left anterior descending artery , can be easily accomplished through coronary angiography , which is not feasible in small animals because of smaller vessels . however , it may cause potential and inevitable injuries to other organs , such as the brain and kidneys , because the remaining microspheres in the left ventricle go into the systemic circulation with blood flow after opening the aorta . hence , it was supposed that 9 m/500,000 microspheres were better because fewer microspheres would go into the systemic circulation although the degree of microinfarction in the 9 m/800,000 and 9 m/500,000 groups was similar . first , the sample size was small that may have an undesirable influence on the results . moreover , a full autopsy was not performed for every mouse , which might have provided more comprehensive information to understand the deaths and the degree of embolization of other organs in this study . in conclusion , the present mouse model with appropriate microspheres might promote further studies on coronary embolization compared with complicated and time - consuming big animal models . this study was supported by grants from the national natural science foundation of china ( no . 81570314 , 81200146 , and 81370322 ) , the zhongshan hospital excellent backbone ( no . 2012zsqn12 ) , the new teacher foundation of ministry of education ( no . 20120071120061 ) , the shanghai municipal science and technology commission ( no . 15xd1501100 ) , and the shanghai municipal commission of health and family planning ( no . xbr2013071 and 20134001 ) . this study was supported by grants from the national natural science foundation of china ( no . 81570314 , 81200146 , and 81370322 ) , the zhongshan hospital excellent backbone ( no . 2012zsqn12 ) , the new teacher foundation of ministry of education ( no . 20120071120061 ) , the shanghai municipal science and technology commission ( no . 15xd1501100 ) , and the shanghai municipal commission of health and family planning ( no . xbr2013071 and 20134001 ) .
background : coronary microembolization ( cme ) has been frequently seen in acute coronary syndromes and percutaneous coronary intervention . small animal models are required for further studies of cme related to severe prognosis . this study aimed to explore a new mouse model of cme.methods:the mouse model of cme was established by injecting polystyrene microspheres into the left ventricular chamber during 15-s occlusion of the ascending aorta . based on the average diameter and dosage used , 30 c57bl/6 male mice were randomly divided into five groups ( n = 6 in each ) : 9 m/500,000 , 9 m/800,000 , 17 m/200,000 , 17 m/500,000 , and sham groups . the postoperative survival and performance of the mice were recorded . the mice were sacrificed 3 or 10 days after the surgery . the heart tissues were harvested for hematoxylin and eosin staining and masson trichrome staining to compare the extent of inflammatory cellular infiltration and fibrin deposition among groups and for scanning transmission electron microscopic examinations to see the ultrastructural changes after cme.results:survival analysis demonstrated that the cumulative survival rate of the 17 m/500,000 group was significantly lower than that of the sham group ( 0/6 vs. 6/6 , p = 0.001 ) . the cumulative survival rate of the 17 m/200,000 group was lower than those of the sham and 9 m groups with no statistical difference ( cumulative survival rate of the 17 m/200,000 , 9 m/800,000 , 9 m/500,000 , and sham groups was 4/6 , 5/6 , 6/6 , and 6/6 , respectively ) . the pathological alterations were similar between the 9 m/500,000 and 9 m/800,000 groups . the extent of inflammatory cellular infiltration and fibrin deposition was more severe in the 17 m/200,000 group than in the 9 m/500,000 and 9 m/800,000 groups 3 and 10 days after the surgery . scanning transmission electron microscopic examinations revealed platelet aggregation and adhesion , microthrombi formation , and changes in cardiomyocytes.conclusion:the injection of 500,000 polystyrene microspheres at an average diameter of 9 m is proved to be appropriate for the mouse model of cme based on the general conditions , postoperative survival rates , and pathological changes .
You are an expert at summarizing long articles. Proceed to summarize the following text: any deviated behavior from the normal will be punished or may be kept behind bars depending upon the crime committed ; sometimes , the crime conducted may also go unnoticed too . studies on such abnormal behavior of human beings are carried out to know the reason for the criminal mind but until date many have ended up with very few definitive results . in this context , the association of behavior of a person and sex chromosomal anomalies has become an exciting challenge for the researchers . sex chromosomal abnormality and its association with psychopath are not only of academic interest but also aim at providing justice to the individual who have been determined as convicts . before the 19 century , discussion of crime was conducted entirely on moral and philosophical terms . it was only in 1876 , an italian anthropologist cesar lombroso published his theories of criminal behavior with scientific approach to understand the criminal behavior of a person . he also stated physiological theories of criminality focusing on person 's physical form as a mark of criminality . in 1949 , sheldon advanced lombroso theory and suggested that some crime might be attributed to a chromosomal abnormality . eventually evidence - based studies established that xyy men are more in offender population when compared with general population . syndrome where men with this condition were thought to be overly aggressive and more likely to become criminals . similarly , in one of the study it was reported that approximately 1% of all individuals institutionalized with mental retardation had an xxy karyotype and approximately half of all mental retardation seen in males originates from a defective gene on the x - chromosome . while the fragile - x site has been implicated in most x - linked mental retardation disorders , other regions of the x - chromosome thus , genes involved in human cognition reside on the x - chromosome . as the syndrome is associated with extra x chromosome , these patients will show positive sex chromatin material in epithelial cells similar to normal females which are termed as barr bodies ( bb ) . the presence of an extra x - chromosome in males is suggestive of klinefelters syndrome ( ks ) , which affects 167/100,000 men . individuals with ks generally presents with severe personality disorders , neuroses and psychoses which are usually in the form of paranoid states and schizophrenic reactions . a study was conducted on ks males as a genetic model for psychotic disorders on ks males and compared with an equal number of normal males followed by an examination of brain mri of these individuals . ten out of eleven ks males had psychiatric disturbance , four of whom had auditory hallucinations compared with normal , where none of the normal individuals showed any abnormality . bartholomew studied psychopath , sex chromosome abnormalities and criminal law and stated that multiple x syndromes in men especially in ks showed inadequate behavior when compared with large control hospital group and also showed aggressiveness in behavior which may include criminality . to examine the crime characteristics of men , we focused on determination of extra x - chromosome in male jail inmates ( xxy syndrome ) and normal healthy male individuals . thus , the present study was concentrated on the determination of extra x - chromosome in jail inmates using epithelial cells from buccal smears ( bs ) and neutrophils in peripheral blood smears ( pbs ) , to detect bb and correlate the same with criminal behavior among jail inmates . this study was performed on male jail inmates and comparison was carried out with equal number of the normal healthy males or controls without any criminal background . a total of 100 male subjects ( fifty jail inmates from central jail and fifty controls ) of above 18 years of age were considered for the study . clearance was obtained from the ethical committee of the institute to conduct a study on jail inmates as well from controls , and written consent was taken from the higher authorities of central jail . a detailed written consent explaining the need for the study was obtained from the jail inmates and control group . to avoid error , the crime history of the jail inmates 46 had committed murder , two were convicted for sexual abuse and remaining two had been convicted of robbery . the identification of the extra x - chromosome was carried out in the study by detecting the presence of bbs in pbs and bs . pbs was prepared by obtaining blood by pricking the finger ( capillary blood ) with all aspetic precautions . pbs and bs were stained using leishman 's stain and cresyl violet stain , respectively . a total of 100 cells were counted in each smear at 100 magnification for the detection of the bbs . in pbs , neutrophils were analyzed for bb as they are present on the nuclear lobes of the neutrophils as drumstick shaped structure [ figure 1 ] . in the case of bss , bbs were found to be attached to the nuclear membrane of epithelial cells [ figure 2 ] . statistical analysis was performed using t - test and mean number of bb was calculated using chi - square test and results were tabulated . photomicrograph showing barr bodies attached to nuclear lobe of neutrophil in peripheral blood smears ( leishman 's stain , 1000 ) the presence of the barr bodies attached to the nuclear membrane of the epithelial cells of buccal smears ( cresyl violet stain , 1000 ) a total of 100 male subjects ( fifty jail inmates from central jail and fifty controls ) of above 18 years of age were considered for the study . clearance was obtained from the ethical committee of the institute to conduct a study on jail inmates as well from controls , and written consent was taken from the higher authorities of central jail . a detailed written consent explaining the need for the study was obtained from the jail inmates and control group . to avoid error , the crime history of the jail inmates 46 had committed murder , two were convicted for sexual abuse and remaining two had been convicted of robbery . the identification of the extra x - chromosome was carried out in the study by detecting the presence of bbs in pbs and bs . pbs was prepared by obtaining blood by pricking the finger ( capillary blood ) with all aspetic precautions . pbs and bs were stained using leishman 's stain and cresyl violet stain , respectively . a total of 100 cells were counted in each smear at 100 magnification for the detection of the bbs . in pbs , neutrophils were analyzed for bb as they are present on the nuclear lobes of the neutrophils as drumstick shaped structure [ figure 1 ] . in the case of bss , bbs were found to be attached to the nuclear membrane of epithelial cells [ figure 2 ] . statistical analysis was performed using t - test and mean number of bb was calculated using chi - square test and results were tabulated . photomicrograph showing barr bodies attached to nuclear lobe of neutrophil in peripheral blood smears ( leishman 's stain , 1000 ) the presence of the barr bodies attached to the nuclear membrane of the epithelial cells of buccal smears ( cresyl violet stain , 1000 ) smears prepared from peripheral blood , revealed positivity in thirty jail inmates for the presence of bb out of total fifty cases which contributed to an overall accuracy of 60% . whereas , in remaining 20 ( 40% ) jail inmates pbs were negative for the same . similarly , detection of bbs was carried out in bss stained with cresyl violet stain in fifty jail inmates . which , revealed positivity for bbs in 18 jail inmates ( 36% ) and depicting negativity in 32 ( 64% ) . the average number of bbs present per high power field in case of pbs of jail inmates were around 26 cells while ; it was reduced to 13 cells per high power field while studying bss [ table 1 ] . positivity for barr bodies in peripheral blood smears and buccal smears of jail inmates similarly , the analysis performed on control group showed a decreased number of positivity for the presence of bbs in pbs contributing around 7 controls ( 14% ) , whereas none of the bss depicted the presence of bbs in controls . the average number of bbs in pbs of controls was 0.32 [ table 2 ] . the comparison of the average number of bbs in the case of jail inmates and in controls showed insignificant results . positivity for barr bodies in peripheral blood smears and buccal smears of control group a comparison was performed between jail inmates and controls for detection of bbs using pbs and bs . in the case of pbs , an overall obtained positivity was 60% ( thirty jail inmates ) while for controls it was reduced to only 14% ( 7 controls ) with an overall positivity of 37% [ table 3 ] . comparison for positivity of barr bodies in jail inmates and controls ( peripheral blood smears ) similarly , the comparison between jail inmates and controls for detecting bbs using bs yielded 36% positivity in jail inmates ( 18 subjects ) , and none were positive in case of controls . the comparison between jail inmates and controls for detection of bb using bs showed statistically significant results [ table 4 ] . comparison for the positivity of barr bodies in jail inmates and controls ( buccal smears ) ks is a genetic disorder which results due to the presence of extra x - chromosome in men . many times ks goes undiagnosed or is diagnosed late due to its varied clinical presentation . studies have revealed a correlation between ks and criminal behavior and have determined the presence of 47 chromosomes , xxy disorder by karyotyping which is an expensive method and can not be performed as a routine screening procedure on a large population . xxy men are found in about 0.81% of males hospitalized with schizophrenia , a four - to - five - fold excess over general live birth rates . this may indicate that genes that are overexpressed in the brains of xxy males may also be relevant to schizophrenia and other psychiatric disorders . since the search for genes leading to susceptibility to major psychiatric disorders has produced inconsistent results . the xxy karyotype may serve as a naturally occurring genetic model to provide clues to the inherited abnormalities that cause these disorders in individuals with normal chromosomal numbers . it is now known that the xxy karyotype occurs in 1 in 500 live male births and is the most common type of human chromosome anomaly . unlike chromosomal duplications or translocations on autosomes this mildness is probably due to inactivation of most genes on the extra x - chromosome . however , there is a class of x - chromosome genes that have homologies on the y chromosome and tend to escape the normal extra x - chromosome inactivation process , as do some other x - specific genes . it is thought that the characteristic features of ks originate from genes that escape inactivation , and are expressed in excess . thus , the tall stature , testicular and sex hormone deficiencies , reduction of secondary sex characteristics , and in some cases , breast development in postpubertal klinefelter 's men may be due to expression of x y homologous genes . similarly , behavioral and cognitive symptoms of ks are likely to result from this class of genes as well . it has been suggested that at least one gene or genes in x y homologous regions of the sex chromosomes that escape normal x - inactivation are crucial for language functioning . thus , the aim of this study was to detect the presence of bbs in jail inmates and to co - relate with their criminal behavior . this is the first study to correlate the criminality of jail inmates by detecting their bbs using pbs and bs among the indian population . the study has obtained 60% positivity for bb in jail inmates as compared to normal controls which showed only 14% in pbs . the comparison between the groups was statistically significant . whereas the presence of bb in bs was less compared to pbs , but when it was compared within the group , it was statistically significant . a similar study using karyotyping was conducted by stochholm et al . and showed the presence of 47 , xxy in a register - based cohort study found moderately increased crime rate in men with ks , whereas few other studies found no such correlation between 47 , xxy and increase in crime in affected men . we found good correlation between the presences of bb in jail inmates which further confirms their psychological behavior to commit crime . bb can also be seen as hyper pyknotic , basophilic , intranuclear structure adjacent to the nuclear membrane in the resting stage of a cell in karyotypic female . it can also be seen adjacent to the nucleolus or be free in the nucleoplasm . it resembles the letter v , w , s or x under electron microscope and measures around 0.8 1.1 in its greatest diameters . it is commonly seen in buccal smear , pulp tissue , hair follicle and vaginal smears commonly used for determination of sex . in a smear only 30% of the cells any nuclear stain can be used for the demonstration of the bbs in bs ; most commonly used ones are hematoxylin and eosin ( h and e ) , papanicolau stain , feulgen stains , guard stains , cresyl violet , carbol fuschin and fluorescent stains . in h and e and papanicolau stains , the bacteria stains heavily , hence the bbs are not noticed prominently . fluorescent stains being more confirmatory for the detection of the bbs but is expensive . in this study , cresyl violet was preferred as it stains bbs more prominently with fewer artifacts and is cost - effective . the comparison between two methods used for detection of bb ( pbs and bs ) was carried out . number of cases for the presence of bb was high in pbs method when compared with bs . it is suggested that pbs is the most accurate and reliable one for detection of bb . comparison between number of bb present in the case of jail inmates and normal healthy male groups revealed that there was no much difference between the two groups for the average number of bb but the number of positive cases for bb was less in controls compared to jail inmates . this suggests that probably the number of bb does not play an important role in the determination of extra x - chromosomes . even though presence of extra x - chromosome in men can be diagnosed using various methods but detection of bb in pbs presence of 60% of the bb in pbs in jail inmates has revealed definite association between the presences of extra x - chromosome in men with criminality . it is well accepted that x - chromosome bears around 1100 genes which are responsible for the normal functioning of brain and testes . with the adequate numbers of studies in large group of population one can find out the reason for the criminality in ks . the detection of bb in males and its correlation to criminality has proved that there is an association between genetic alteration and antisocial behavior of a person . in this study , the jail inmates showed increased number of positivity for bb , which is suggestive of extra x - chromosome . this preliminary study revealed presence of bb in jail inmates which can be correlated with their criminal behavior but further confirmation needs to be done for the presence of the extra x - chromosome by performing karyotyping . nevertheless , detection of bb using pbs and bs appears to be a simple , cost - effective and appropriate screening method to detect xxy syndrome in criminals . presence of bb in jail inmates will further confirm their criminal mind , which has been suspected to have committed crime and it also confirms the crime in victims who is already a convict . authors intend to further study and correlate the presence of bb with karyotyping to confirm our preliminary findings .
background : cytogenetic studies from past decades have shown that interphase cells of female cats contain a densely stained chromatin mass in their nuclei called as barr bodies ( bbs ) named after the scientist murray barr . bbs are unique chromatin structures formed due to the condensation of the x - chromosome . many psychopathic disorders originate from defective genes including the multiple x syndromes . males with extra x - chromosome generally present with severe personality disorder . the present study was conducted to determine the presence of extra x - chromosome in male jail inmates through the detection of bb in peripheral blood and buccal smear.materials and methods : study included 100 male subjects ( fifty jail inmates and fifty controls ) , after obtaining the consent , peripheral blood smears ( pbs ) and buccal smears ( bs ) were prepared and stained using leishman 's and cresyl violet stain respectively . one hundred neutrophils in pbs and epithelial cells in bs were screened for detection of the bb ; accumulated data were tabulated and statistically analyzed using t - test and chi - square test.results:60% of cases in pbs and 36% in bs showed positivity for the presence of bb in jail inmates as compared to 14% of cases in pbs and none in bs were positive for bb in controls.conclusion:presence of bb in male suggests increased likelihood of criminal tendencies . further studies are to be carried out to compare the results with karyotyping .
You are an expert at summarizing long articles. Proceed to summarize the following text: oral health is an important component of general health and is associated with the development of a healthy personality . in modern times , much focus is laid upon the development of orofacial disorders and treatment of the resulting malocclusions.1,2 the evaluation of different types of malocclusions existing within a population is important in order to plan orthodontic treatment and determine the resources required for the service.1,3 measuring and recording the severity and prevalence of malocclusion are important as it can be used as an epidemiological tool for preventive procedures and evaluating the occlusal status of subjects in the community and establishing the treatment priority.1,4 it is essential to know the prevalence of malocclusion in any society , as it reveals the true extent of the problem and the general public can be educated , so that appropriate preventive and corrective measures can be instituted . prevalence studies help the specialists in establishing proper preventive and treatment need in a given region.1 the malocclusion can be defined as an occlusion in which there is a mal - relationship between the arches in any of the planes or in which there are anomalies in tooth position beyond the normal limits.5,6 the malocclusion may lead to the individual feeling uncomfortable in social contacts , losing career opportunities and hence being embarrassed by their facial appearance.6,7 the etiology of malocclusion could be genetic or environmental and/or combination of both the factors along with various local factors such as adverse oral habits , tooth anomalies , form and developmental position of teeth.6,8 the malocclusion has been shown to affects oral health , leads to increased prevalence of dental caries and may result in temporo - mandibular joint disorders.5,6 the prevalence of malocclusion varies from country to country and among different age and sex group.6 in saudi arabia , governmental sectors provide free orthodontic treatment for saudi citizens thereby leading to increased burden of work on these health care providers.9 the dental aesthetic index ( dai ) is an orthodontic index used on socially defined aesthetic norms has got regression equation that links the public s perceptions of dental aesthetics with the objective physical measurements of the occlusal traits associated with malocclusion mathematically . it can be used as a practical tool for epidemiologists and other dental personnel for screening orthodontic treatment need.6,10 - 12 hence , the present study was conducted to evaluate the malocclusion in subjects reporting for orthodontic treatment among saudi populations in asser region by using dai . all study participants reporting to king khalid university - college of dentistry , asser central hospital and civil hospital for orthodontic needs were recruited as study samples . informed consent was obtained from all study participants , and ethical clearance was obtained from all the institutions . a pre - designed proforma was used to evaluate malocclusion which consisted - demographic details , angles classification , overjet , overbite , cross bite ( anterior and posterior ) , open bite , scissor bite , spacing , crowding , canine guidance , group guidance and dai . type - iii clinical examination as recommended by american dental association ( ada ) specification was followed . all three examiner involved in the study were calibrated , and inter examiner reliability was calculated using kappa statistics ( = 0.94 ) . data were entered in excel 2010 and was analyzed using statistical package for social sciences 16.0 . the frequency and percentage distribution of the study variables among the study participants was calculated ( graph 1 ) . the association of the age with study variables was assessed using the chi - square test . the mean overbite and dai was tested using one - way analysis of variance ( anova ) among the various age groups . data were entered in excel 2010 and was analyzed using statistical package for social sciences 16.0 . the frequency and percentage distribution of the study variables among the study participants was calculated ( graph 1 ) . the association of the age with study variables was assessed using the chi - square test . the mean overbite and dai was tested using one - way analysis of variance ( anova ) among the various age groups . a total of 162 participants was included in the study with a mean age of 27.07 9.76 years . maximum ( 75% ) of the participants had angle s class i molar relation followed by angle s class ii and iii . table 2 shows the association of the study parameters with age of the study participants using the chi - square test . there was no statistically significant association found between age and any of the study parameters . table 3 shows a comparison of mean over jet and dai score in the various age groups using one - way anova there was a statistically significant difference in the mean dai score between the participants in the various age groups ( p < 0.05 ) ( table 4 and graph 2 ) . comparison of mean over jet and dai score in the various age groups using one - way anova . a large number of indices to assess malocclusion have been developed for the estimation of orthodontic treatment need in particular populations or communities , for patients who can be treated in a certain dental care system and to establish priorities when resources are limited . it is a controversial view as to which individual characteristics and occlusal features should be assessed in order to evaluate the orthodontic treatment need,13 however , the various malocclusion indices used in epidemiological studies of malocclusion in different countries have innumerable similarities and evaluation criteria . such indices include the dai ( 1986)14 and the index of orthodontic treatment need ( 1989).15 the dai links the clinical and esthetic components mathematically in a regression equation to arrive at a single mark , which combines the physical and esthetic aspects of the occlusion using ten evaluation criteria . it is based on a social acceptability scale of occlusal conditions16 and has been used in many studies to determine orthodontic treatment need in different countries.17 - 22 a dai scale that divided the continuous index score defined by the equation into four malocclusion severity levels was established and is readily usable and has been extensively used in orthodontic care programmes or malocclusion prevalence studies.12,23 it was included in the world health organization oral health survey methods ( 1997 ) . the dai is based on dental aesthetics , but is does not include functional considerations or potential risks to the dentition that could be functionally detrimental to the individual , but are not aesthetically significant.12,13,23 the majority of patients who attended for orthodontic treatment were of the younger age groups . these findings were similar to those of al - balkhi and al - zahrani.24 this is consistent with the fact that youngsters are highly concerned about their appearance even though the malocclusion may or may not affect their functional ability . a lower percent of adult patients attended the clinics that may be due to a lower rate of referral of adults for orthodontic care by clinicians as well as the fact that adults are more concerned by the functional rather than the aesthetic aspect of their dentition . the frequency distribution of angle s types of malocclusion among the study samples was in close agreement with that reported by al - balkhi and al - zahrani,24 al - emran et al.25 and nashashibi et al.26 the class i molar relationship type was the most frequently observed , followed by class ii and class iii and it was the most predominant feature of saudi arabian patients attending for orthodontic treatment . almost half the patients attending for orthodontic treatment were found to have crowding or arch length insufficiency . the crowding was more commonly seen in the anterior arch segment than posterior , and this was in agreement with previous studies of the saudi population.24 - 26 nevertheless , the percentage of crowding in this study was higher . the dental spacing was similar to those reported by al - balkhi and al - zahrani,24 al - emran et al.25 and nashashibi et al.26 in saudi arabia , governmental sectors provide free orthodontic treatment for saudi citizens thereby leading to increased burden of work on these health care providers.9 the planning orthodontic services and estimating the required resources and manpower requires meticulous evaluation of self - perceived and normative need for orthodontic treatment as well as other factors affecting these needs such as social , demographic , and psychosocial factors.24,27 unnecessary referrals by general practitioners increase the burden on the healthcare system and lengthy waiting lists for orthodontic treatment can be eliminated by limiting free treatment to patients only with severe malocclusions that affect the patient s function and aesthetics.28,29 it may also help to predict patients level of interest and motivation toward the orthodontic treatment , create awareness by planning educational programs in schools . it can also influence media to increase patient s awareness and to overcome obstacles and barriers in seeking treatment.9,30 the limitations of the present study include a small sample size and lack of a comparison group . further studies are recommended to include a larger sample size with various socio - demographic being taken into consideration as well as comparison studies with various populations across the world . knowing the prevalence of malocclusion in a community is of utmost importance so that appropriate steps can be taken to prevent and treat the dentofacial irregularities . malocclusion is prevalent in saudi arabia and is a serious concern to the dental fraternity .
background : oral health is an important component of general health and is associated with the development of a healthy personality . the malocclusion has been shown to affects oral health , leads to increased prevalence of dental caries and may result in temporo - mandibular joint disorders . hence , the present study was conducted to evaluate the malocclusion in subjects reporting for orthodontic treatment among saudi population in asser region by using dental aesthetic index ( dai).materials and methods : a cross - sectional survey was designed . a total of 162 study samples was participated in the study . a pre - designed proforma was used to evaluate various parameters determining malocclusion and dai was used.results:the mean age of 27.07 9.76 years . the mean dai score of the study participants was 27.27 13.83 . maximum ( 75% ) of the participants had angle s class i molar relation followed by angle s class ii and ii . comparison of mean over jet and dai score in the various age groups using one - way anova there was a statistically significant difference in the mean dai score between the participants in the various age groups.conclusion:knowing the prevalence of malocclusion in a community is of utmost importance , so that appropriate steps can be taken to prevent and treat the dentofacial irregularities .
You are an expert at summarizing long articles. Proceed to summarize the following text: the mammalian nonreceptor tyrosine kinases ( nrtks ) are divided into ten families : src , abl , jak , ack , csk , fak , fes , frk , tec , and syk . in addition to their tyrosine kinase catalytic domains , they all contain noncatalytic domains that are important in enzyme regulation and substrate recognition . like all eukaryotic protein kinases , nrtk catalytic domains have an n - terminal lobe ( n - lobe ) that contacts atp , and a larger c - terminal lobe ( c - lobe ) . the activation state of the tyrosine kinases depends on the orientation of an alpha helix ( c ) located in the n - lobe . in the active conformation , the c helix projects inward toward the atp - binding site . the conformation of a flexible segment in the c - lobe ( the activation loop ) also has a key role in the regulation of the enzyme activity . the regulatory importance of the phosphorylation of the activation loop varies in the different families of nrtks . for instance , src activation is strongly dependent on autophosphorylation , while csk lacks the autophosphorylation site . the contributions of the noncatalytic domains to enzyme regulation are well understood in the case of the src - family of nrtks . these enzymes have an n - terminal myristoylation site followed by an sh3 domain , an sh2 domain , and a kinase domain . two tyrosine residues are important for their activity : tyr 416 in the activation loop and tyr 527 in the c - terminal tail . structural studies showed the spatial arrangement of src domains in the inactive state , in which the noncatalytic domains mediate intramolecular interactions that maintain the enzyme in a closed , inactive conformation . in this conformation , the sh3 domain is bound to a proline - rich helix located between the sh2 domain and the kinase domain , and the sh2 domain is bound to the phosphorylated tyr 527 [ 7 , 8 ] . activating signals provided by protein - protein interactions that destabilize these intramolecular interactions may be of three types : the binding of the sh3 domain to a proline - rich motif ( sh3 binding domain ) ; the dephosphorylation of the tyr527 in the c - terminal tail ; the binding of the sh2 domain to other phospho - tyr containing proteins . in addition , the noncatalytic domains provide surfaces for different types of interactions that couple the activation of the catalytic domain to substrate recognition [ 2 , 12 , 13 ] . for example , abl kinases have similar arrangements of domains as src - family kinases , but the regulatory mechanisms are different ; this has important implications for the design of pharmacological inhibitors . in both src- and abl - family nrtks , the sh3 domain interacts with a poly - proline helix in the linker region between the sh2 domain . in contrast to src , the sh2 domain of abl interacts in a phospho - tyr independent manner with the c - terminal lobe of the kinase domain . an additional difference is that an n - terminal cap and a myristoyl group wrap around the base of the kinase domain of abl and stabilize the inhibited structure . importantly , the conformation of the inhibited activation loop is unique to the c - abl catalytic domain . the inhibitor imatinib ( gleevec ) has been particularly successful due to its ability to target this conformation . as new structures of nrtks structural studies have illuminated the contributions of noncatalytic domains to the regulation of syk- [ 3 , 5 ] , csk- [ 6 , 1517 ] , and fes - family kinases . for instance , in csk and fes kinases , interactions between the sh2 and the catalytic domains stabilize the active conformation . in general , the noncatalytic domains of all nrtks the domain architecture of human ack1 ( also called tnk2 ) is shown in figure 1 ( top ) . ack1 is a 120 kda protein with an n - terminal sterile alpha motif ( sam ) domain , a kinase domain , an sh3 domain , and a cdc42/rac - interactive domain ( crib ) . several functionally relevant regions have been identified in the c - terminal region of ack1 : multiple proline - rich sequences , a clathrin - binding motif , an ubiquitin binding domain , and a region that shares a high homology with mig6 . they are the only nrtks with the sh3 domain located c - terminal to the kinase domain , and they are also the only tyrosine kinases with a crib domain . in view of its unique domain composition , the regulatory features of ack1 are likely to differ from those observed in other nrtks . ack1 belongs to a family of nrtks that includes human tnk1 , and homologous proteins in mouse , cow , drosophila melanogaster , and caenorhabditis elegans [ 23 , 24 ] . although all the members share the overall domain architecture shown in figure 1 ( i.e. , kinase domain and sh3 domain ) , there are major differences in the c - terminal regions of the proteins . furthermore , tnk1 , dack ( from drosophila ) , and kos1 ( from mouse ) lack crib domains . ack1 , the first member of the ack family , was cloned from a human hippocampal expression library by its ability to bind gtp - bound cdc42 specifically and not rac1 or rhoa . the gene encoding ack1 is located on chromosome 3q29 in humans , a region that is associated with recurrence of prostate cancer and is a predictor of metastatic relapse in breast cancer . tnk1 ( thirty - eight - negative kinase 1 ) was originally cloned from hematopoietic stem / progenitor cells from umbilical cord blood in humans . tnk1 is a member of the ack family with a predicted size of 72 kda and the same domain architecture as ack for the region common to both family members . in contrast with the other members of the ack family ( namely , ack1/tnk2 , ack2 , and dpr2 ) , but similarly to dack , tnk1 , and kos1 lack the crib domain [ 24 , 27 ] . tnk1 , was found to be specifically expressed in umbilical blood and not other hematopoietic tissues and to interact with phospholipase c-1 through its proline - rich region . it has been reported that during embryonic development , tnk1 blocks nf-b activation and thereby promotes apoptosis mediated by the tnf signaling pathway . kos1 is a 47 kda protein that was cloned from differentiating murine embryonic stem cells . the kos1 gene is located in chromosome 11 in mice and it is thought to be a murine splice variant of the human tnk1 . ack2 has a predicted size of 83 kda and contains the same basic domain structure as ack1 . ack2 is thought to be an isoform of ack1 that is generated by alternative splicing and is not encoded by a different gene . the ack2 c - terminal region contains a 15-residue insert and has 344 fewer amino acids than ack1 . in humans there is no evidence for the existence of an ack2 homologue [ 18 , 25 ] . however , a number of the experimental studies on ack ( activation pathways , biochemical properties , and biological roles ) focused on ack2 , and they will be discussed throughout this paper . in drosophila the roles of dpr2 and dack have not been dissected but they are thought to be different because dpr2 has a cdc42-binding domain , while dack lacks one . the tyrosine kinase activity of dack has been implicated downstream of cdc42 in the pathway that leads to dorsal closure of the epidermis during embryogenesis . in c. elegans , ark-1 ( a ras - regulating kinase 1 ) , a cytoplasmic tyrosine kinase that is related to ack1 , acts as a negative regulator of egfr signaling . in a yeast two - hybrid analysis , ark-1 interacts with the grb2 homologue , sem5 . the degree of conservation of the full - length sequences varies among the family members . the common core that is conserved in all of the ack family members spans from the n - terminus to the sh3 domain . the amino acid identity of this core ( compared with ack1 ) ranges between 100% for ack2 and 29% for kos1 ( figure 1 ) . the ack1 kinase domain itself shares 40% amino acid identity with other nonreceptor and receptor tyrosine kinases . phylogenetic analyses of human tyrosine kinase catalytic domains place ack1 near epidermal growth factor receptor ( egfr ) family kinases [ 32 , 33 ] . as described below , ack1 and egfr may also share aspects of enzymatic regulation . expression of ack1 has been demonstrated in several tissues in mammals , with the highest expression in spleen , thymus , and brain . a number of stimuli , including egf , pdgf , bradykinin , agonists of the m3 muscarinic receptor , and integrin - mediated cell adhesion [ 35 , 36 ] promote phosphorylation and activation of ack1 . upon egf stimulation , ack1 phosphorylates and interacts with the rho - family gtpase cdc42 and the adaptor protein grb2 and these interactions are essential for this pathway . the association of ack1 with grb2 has been shown in several studies through different approaches . in vitro studies showed that ack1 associates with several sh3 domains , including the sh3 domain from grb2 . ack1 association with multiple receptor tyrosine kinases such as axl , ltk , and alk is also dependent on grb2 . ack is involved in integrin 1-mediated signaling pathways that modulate processes such as cell migration , cell adhesion , and cell spreading . ack2 is present in a complex with integrin 1 ; it is activated by cell adhesion to a fibronectin - coated surface in a cdc42-dependent manner and it activates the phosphorylation of jnk . in response to cdc42 activation , ack1 mediates the phosphorylation of p130cas to promote cell migration [ 35 , 40 ] . ack1 is found in a complex with cas , crk , and cdc42 in collagen - stimulated cells . a signaling complex composed of cdc42 , ack1 , and cas also participates in the regulation of melanoma cell spreading by melanoma chondroitin sulphate proteoglycan ( mcsp ) , a proteoglycan that is expressed on the surface of melanoma cells . the expression of ack2 in hela cells increases cell migration in a mechanism that also involves p130cas and crkii . thus , one of the physiological functions of ack1 appears to be related to the regulation of cell adhesion and cell migration . these studies also suggest that ack1 activity is involved in the development of metastatic potential of transformed cells . several studies have focused on the role of ack1 in egf receptor trafficking and dynamics . egfr stability and recycling are regulated by interactions between ack1 and multiple protein partners including ubiquitin , clathrin heavy chain [ 20 , 42 ] , and sh3px1 [ 43 , 44 ] . in hela cells , overexpression of ack1 inhibits egfr endocytosis and causes the accumulation of egf in internal structures of endocytic origin , suggesting that ack1 is involved in the intracellular sorting of activated egfr . the knock - down of ack1 increases the rate of recycling i - egf to the plasma membrane and reduces the degradation of i - egf , suggesting that ack1 promotes degradation of egfr . in cos7 cells , overexpression of ack1 promotes egfr degradation by binding to the ubiquitinated egf receptor . increased stability of ack1 caused by a somatic mutation ( s985n ) in the ubiquitin binding domain ( uba ) results in defective egfr downregulation and sustained activation of downstream signaling . in addition to the effect of ack1 on receptor stability and dynamics , there is a reciprocal effect on ack1 stability . after receptor stimulation by egf , ack1 is recruited to an egfr complex [ 21 , 47 ] . this complex promotes the activation of ack1 and egfr degradation [ 21 , 45 , 46 , 48 ] as well as ack1 turnover [ 49 , 50 ] . the egf - induced degradation of ack1 is signaled by ubiquitination [ 49 , 50 ] . , the e3 ubiquitin ligase nedd4 - 2 was shown to ubiquitinate the uba domain of ack1 and induce its degradation by proteasomes . in a second report , the e3 ligase nedd4 - 1 was demonstrated to ubiquitinate the ack1 sam domain , while the deletion of the uba domain enhanced ubiquitination . in these experiments , the degradation of ack1 was mediated by lysosomes rather than by proteasomes . in both cases , the ww domains of the e3 ligases bound to a conserved ppxy motif in ack1 . the crystal structure of the isolated kinase domain of ack1 has been solved in both phosphorylated and unphosphorylated forms . in these structures , the activation loop conformations are similar and do not occlude the substrate - binding site , suggesting that the phosphorylation of the activation loop may not play a dramatic stimulatory role . the only other structural information available is for the isolated crib domain of ack1 in a complex with cdc42 . in the absence of a structure of full - length ack1 , biochemical experiments have helped to reveal how each of the domains contributes to ack1 function . a major autophosphorylation site has been mapped to the y284 in the activation loop . a construct containing residues 1- 476 , corresponding to the n - terminus , the kinase domain , the sh3 domain , and the crib domain ( nt - kinase - sh3-crib ) is the longest construct that has been purified to homogeneity . this construct is activated , although modestly ( 3-fold ) , by the phosphorylation of y284 . transformed cell lines , y826 , y856 , and y857 of ack1 are phosphorylated ( figure 2 ) . the phosphorylation of y826 was also identified in a study of phosphorylated proteins downstream of insulin signaling . y857 of ack1 was identified in a mass spectrometry study in which protein phosphorylation events downstream of the egfr - family member her2 were analyzed . in addition , the phosphorylation of y518 was identified in a study of global tyrosine phosphorylation patterns in cancer cells . interestingly , y826 , y857 , and y858 are located in the mig6-homology region ( mhr ) , a portion of ack1 that participates in inhibitory intramolecular interactions . it is possible that the mhr of ack1 is a substrate for oncogenic bcr - abl , or for insulin and egf receptors , resulting the release of autoinhibition . relatively little information is available about the regulatory mechanisms of ack1 and the roles that each domain plays in ack1 regulation . the purified nt - kinase - sh3-crib polypeptide is active in vitro and can be used in enzymatic assays . the peptide substrates that are the most efficiently phosphorylated by this construct are eaiyaapfakkkg ( abl consensus substrate ) or kviydfiekkkkg ( peptide derived from wasp tyrosine phosphorylation site ) . in addition to this tyrosine kinase activity , ack1 acts as a dual - specificity kinase towards wasp and the phosphorylation of a serine residue of wasp ( wiskott - aldrich syndrome protein ) increases the ability of wasp to polymerize actin . ligands for the sh3 domain and the crib domain bind to this construct , but fail to activate it , although addition of cdc42 in vivo does increase ack1 y284 phosphorylation . residues important for the interaction between ack1 and cdc42 have been identified from the nmr structure of the complex formed by a peptide spanning the residues 548489 of ack1 ( crib domain is 548475 ) and cdc42 . one way of studying the role of each domain in ack1 regulation is to assess the effects of point mutations on the enzyme 's phosphorylation state ( figure 2 ) . a mutation that occurs in ovarian endometrioid carcinoma ( e346k ) has been helpful to gain insight into the role of the mig6 homology region ( mhr ) . these studies have suggested that there is an autoinhibitory interaction between the c - lobe of ack1 kinase domain and the mhr . mutations such as v365r ( in the kinase domain ) or f820a and e346k ( in the mhr ) destabilize this conformation and activate ack1 . ack is activated by additional cancer - associated mutations located in the sam domain ( r34l and r99q ) , and in the sh3 domain ( m409i ) , but the mechanism is not clear . several studies have produced a constitutively active ack1 by the point mutation of l487f in the crib domain [ 56 , 57 ] . the position of this mutation is thought to be analogous to l107f in pak1 , which is known to prevent an autoinhibitory interaction . however , it is not known whether the crib domain of ack1 participates in autoinhibition . a mutation designed to prevent the binding of the sh3 domain to its ligand ( w426k ) was reported to produce activation of ack1 . in contrast , a point mutation that disrupts cdc42 binding ( h464d ) reduced ack1 autophosphorylation . the deletion of the n - terminal sam domain reduced ack1 autophosphorylation and kinase activity [ 18 , 64 ] . the sam domain has the potential to drive ack1 to the plasma membrane [ 18 , 64 ] and to form dimers or multimers that increase the local concentration in order to stimulate ack1 activation . interestingly , the deletion of the ack1 sam domain also prevented egf - induced ack1 ubiquitination , consistent with the requirement of the sam domain for activation and with the observation that ack1 is ubiquitinated after activation . these results suggest that the individual domains of ack1 are involved in regulatory interactions that could be either inter- or intramolecular . the downregulated state of ack1 involves the intramolecular interaction of the mhr with the kinase domain ( figure 3(a ) ) . as a result of a stimulatory signal , provided by activated egfr , one or more tyrosine residues located in the ack1 mhr are phosphorylated ( figure 3(b ) ) . in addition , the sam domain of ack1 is required for full autophosphorylation and activation of ack1 . it is not clear whether the sam domain participates in autoinhibition but as noted above , it is involved in subcellular targeting and required for maximal enzymatic activity . the domain composition of ack1 is complex and it is likely that in the future , additional interactions ( both intra - and intermolecular ) will be described that will explain in more detail the molecular mechanisms of ack1 regulation and signaling . ack1 has been implicated in several stages and several types of cancer ; this topic has been reviewed recently in . early experiments demonstrated that a peptide derived from the ack1 crib domain blocked the ras - mediated transformation of nih-3t3 cells . ack1 expression was later shown to be required for the survival of cells transformed by v - ras . in a study of ~180 advanced - stage cancers , 9% of the lung tumors and 14% of the ovary tumors had an amplification of the ack1 gene on chromosome 3 . in addition , 42% of the aggressive lung tumors and 77% of metastatic hormone refractory prostate tumors showed overexpression of the ack1 mrna . ack1 overexpression enhanced migration of human mammary epithelial cells ( hmecs ) and increased metastasis in the mouse mammary 4t1 breast cancer system . moreover , activation of ack1 by overexpression of ack1 produced several epithelial - to - mesenchymal transition ( emt ) phenotypes in the human breast cancer cell line mda - mb-231 . in renal cancer cells , activation of ack1 by a somatic point mutation also resulted in emt phenotypes . thus , activation of ack1 is associated with poor prognosis and metastatic phenotypes in human tumors . in addition , the knockdown of ack1 in breast cancer cells inhibits cell migration . this suggests a role for ack1 as a metastasis determinant , perhaps as a modifier of other pre - metastatic lesions . the mechanistic basis of ack1 's involvement in prostate tumorigenesis has been explored in detail . in the cultured prostate cancer cell line lncap , the androgen receptor ( ar ) is a transcriptional activator that plays important roles in the development of advanced stage ( androgen - independent ) prostate cancer . activated ack1 has been shown to phosphorylate and activate ar function and to promote the progression of prostate cancer . in addition , ack1 tyr284 phosphorylation was recently found to correlate with disease progression and proposed to be prognostic of progression of prostate cancer . a large - scale study that screened cancer tissues for somatic mutations identified four missense mutations in ack1 . in lung adenocarcinoma and ovarian carcinoma , two mutations located in the n - terminus ( r34l and r99q , resp . ) were identified ; in ovarian endometrioid carcinoma , a mutation in the kinase catalytic domain ( e346k ) was identified ; and in lung adenocarcinoma , a mutation in the sh3 domain ( m409i ) was found ( figure 2 ) . the four cancer - associated mutations activate ack1 and promote anchorage independent growth and cell migration . an additional somatic mutation found in renal cancer , s985n , located in the uba domain of ack1 , increases ack1 stability and enhances oncogenic signaling through egfr . the functions of ack1 in cancer cells appear to be of two types : kinase - independent and kinase - dependent . the effect of ack1 on cell proliferation is independent of ack1 activity . in a phosphoproteomic study of the action of the antitumor drug dasatinib in lung cancer , this study highlighted a possible scaffolding function of ack1 in the context of control of cell growth . in contrast , the activation state of ack1 appears to be important in processes that involve enhanced cell motility , that are crucial in the metastatic stage of cancer progression and correlate with poor prognosis [ 25 , 58 , 67 ] . in breast cancer , ack1 phosphorylates and promotes the activation of akt , an important mediator of signaling pathways that lead to transformation . a number of ack1 substrates have been identified in normal cells as well as cancer cells . these results , together with the enhanced migration of cells overexpressing ack1 or expressing an activated form of ack1 , suggest that ack1 plays a role in the regulation of cell adhesion and migration . ack1 also phosphorylates androgen receptor ( ar ) , increasing the levels of phosphorylated ar in prostate cancer and promoting ar - mediated gene transcription [ 68 , 69 ] . a comprehensive list of known ack1 substrates and interacting proteins was recently published , and it includes diverse molecules involved in signaling such as receptor tyrosine kinases ( merk , egfr , pdgfr , axl , alk , and ltk ) , membrane proteins ( integrin , mcsp ) , adaptor molecules ( grb2 , hsh2 ) , nucleotide exchange factors ( dbl ) , a transcription activator ( ar ) , a tumor suppressor ( wwox ) , and a proto - oncogene ( akt / pkb ) . other ack1 interactors include proteins involved with vesicle dynamics ( clathrin , snx9 ) and cytoskeleton remodeling ( wasp ) . a number of sh3 containing proteins , such as hck , grb2 , and snx9 [ 44 , 72 ] have been identified as ligands for the proline rich region of ack1 . recently , the e3 ubiquitin ligases nedd4 - 1 and nedd4 - 2 were shown to bind to a ppxy motif of ack1 and to regulate its proteolytic degradation [ 49 , 50 ] . nonreceptor tyrosine kinases share several common features . the nrtk catalytic domains never occur in isolation ; they are tethered to noncatalytic modular domains that play important roles in enzymatic function . the biochemical properties of the individual noncatalytic domains are similar to those found in other signaling proteins . however , the regulatory mechanisms are diverse and vary between the different families of nrtks . to date , 15 out of the 32 nrtks in the human genome have been shown to have oncogenic potential . activation of ack1 ( by overexpression or mutation ) promotes migration and emt and correlates with cancer progression . thus , ack1 has emerged as a candidate for anti cancer drug design ; ack1 inhibitors could potentially be used in combination therapies with inhibitors of other tyrosine kinases , such as egfr . knowledge about nrtk regulatory mechanisms can be exploited for the development of specific inhibitory drugs . for example , imatinib inhibits abl ( but not src ) because it binds to a specific conformation of abl that src is not capable of adopting . moreover , the inhibited conformation of abl appears to be the result of the interplay of the noncatalytic domains . ack1 is regulated by a novel mechanism that shares basic principles with other nrtks but also has unique features dictated by its unusual domain structure . investigations into the regulatory mechanisms and conformational states of ack1 will be an important prelude to the development of specific ack1 inhibitors .
ack family non - receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties . human ack1 ( activated cdc42-associated kinase ) is ubiquitously expressed and is activated by signals that include growth factors and integrin - mediated cell adhesion . stimulation leads to ack1 autophosphorylation and to phosphorylation of additional residues in the c - terminus . the n - terminal sam domain is required for full activation . ack1 exerts some of its effects via protein - protein interactions that are independent of its kinase activity . in the basal state , ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the c - terminal region . inappropriate ack1 activation and signaling has been implicated in the development , progression , and metastasis of several forms of cancer . thus , there is increasing interest in ack1 as a drug target , and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design .
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Proceed to summarize the following text: tobacco smoke is a complex mixture of more than 4500 chemicals , many of which have toxic and/or carcinogenic activity . some of the components , which could be in the form of gases , vapors , and particulates , include carbon monoxide , hydrogen cyanide , phenols , acrolein , ammonia , formaldehyde , nicotine , nitrosamine , tar , heavy metals , and at least 48 known cancer - producing substances . cigarette smoking is a worldwide social epidemic and it is one of the main causes of preventable death and disability . it is an established risk factor for premature mortality due to cancer , cardiovascular disease , and chronic obstructive pulmonary disease . the increased susceptibility of cigarette smokers to infections reflects multifunctional alteration of their innate and adaptive immune responses . acrolein , a toxic unsaturated aldehyde , affects neutrophil functions and thus increases susceptibility to lung infections . leukocytosis is a well - known effect of cigarette smoking though the function of these cells is greatly reduced . reported that a single cigarette provided enough toxic material that completely inhibited the function of oral salivary neutrophils in situ . reduced phagocytic activity of neutrophils was also reported in smokers , which could be responsible for decreased defence of the gingival against bacterial attack . continued exposure to cigarette smoking has been shown to affect both humoral and cellular immune responses . initially ( hours to days ) , there is an acute depression of the immune response followed by stimulation ( weeks to months ) , and finally depression of the immune system sets in . this causes a decreased response to antigens and reduced serum concentration of igg , igm and iga , and also increased levels of autoantibodies notably ; anti - nuclear rheumatoid factors . although several reports have shown that cigarette smoking significantly reduces serum levels of immunoglobin classes in humans , a recent report by arinola et al . similarly , conflicting observations have been reported on levels of salivary immunoglobulin classes in smokers . bennet and read reported significantly reduced level of salivary iga in smokers while engstrm and engstrm reported increased levels of salivary iga in smokers . more so , research on salivary levels of immunoglobulin classes in cigarette smokers has been rarely explored . to explore , for the first time among nigerians , the interplay between components of cigarette smoke and salivary levels of immunoglobulin classes , our study estimated salivary immunoglobulin classes ( igg , iga , igm , ige ) in nigerian smokers to provide oral immunological based reasons for oral diseases in cigarette smokers . forty - five ( 45 ) subjects were recruited for this study after obtaining informed consent from each subject and an ethical approval from the university of ibadan / university college hospital ( u.i / u.c.h ) joint ethics review committee . the test group consisted of 24 active smokers who smoke at least 6 sticks of cigarette per day while the control group comprised 21 sex- and age - matched non - smokers who were apparently healthy . also excluded were individuals with pregnancy , diabetes , and human immunodeficiency virus ( hiv ) infection . a short structured questionnaire was administered on each subject to obtain information on age , sex , occupation , cigarette smoking , and drug consumption . about 5 ml of unstimulated saliva was collected from each subject using the spitting method into plain sample bottles . the samples were collected between 9 am and 11 am , at least 1 h after eating or washing of mouth . the samples were centrifuged at 3000 g for 5 min and the clear supernatant gently pipetted out into another clean plain bottle and stored at -20c until analysed . immunoglobulin levels were estimated using enzyme - linked immunosorbent assay ( elisa ) supplied by immunology consultant laboratory , usa . ige kit was supplied by leinco technologies , usa . the assay was carried out following manufacturer 's instructions . student 's t - test ( unpaired ) was used to determine significant difference between the means . student 's t - test ( unpaired ) was used to determine significant difference between the means . the mean ages of smokers and non - smokers were 39.9 and 39.5 years , respectively . no significant differences were observed in the mean salivary levels of igg , iga and ige . only igm was significantly low in smokers compared with non - smokers ( p = 0.038 ) . the mean level of salivary ige was lower in smokers compared with control . only 1 smoker ( 4.17% ) had a detectable level of salivary ige ( 0.04 ) while two non - smokers ( 9.52% ) had detectable levels of ige ( 0.24 ) . also the proportion of smokers with detectable level of salivary ige was lower compared with controls [ table 1 ] . cigarette smoking is among social practices commonly found in some nigerian youth , despite its adverse health consequences . gingivitis , periodontitis , pocket depth , attachment loss , alveolar bone loss , and tooth loss are some of oral pathologies commonly found in cigarette smokers . herr et al . reported that current or former smoking is associated with reduced levels of human -defensins 2 ( hbd2 ) in pharyngeal washes and sputum of patients with acute pneumonia . of note , smoking is associated with reduced levels of surfactant proteins a and d ( sp - a and sp - d ) . its increase could be due to increased local infection , increased antigenic inflammatory stimulus , increased local synthesis , and local host reaction against the presence of disease . levels of other immunoglobulin classes ( igg , igm and ige ) have also been reported to decrease in oral diseases as observed in gingival fluid exudates . the mean levels of all the immunoglobulin classes were reduced in the saliva of smokers compared with non - smokers . however , only the salivary igm mean level was significantly reduced in smokers ( p = 0.038 ) . this observation contradicts the report of engstrm and engstrm who observed increased salivary iga only . they suggested that their observation could be a reflection of protection of the oral mucosa . earlier report by bennet and read who reported a significantly low salivary level of iga only partially supports our observed low salivary iga level in smokers . in our study , the exclusion of subjects with oral diseases could be responsible for the observed differences in salivary iga as there could be upsurge in oral antibodies production consequent to oral infection or diseases . more so , our observation could be as a result of nicotine contained in cigarette as nicotine affects the exocrine glands by an initial increase in salivary secretions followed by inhibition of the secretions . a study carried out on patients with oral mucosal disease showed higher level of salivary igg . the causative effect was suggested to be increased permeability of oral mucosa which made it easy for the passage of igg from vascular and extra vascular compartment into saliva by passive transmucosal diffusion . experimental studies also showed that mice that were chronically exposed to cigarette smoke were more susceptible to influenza and murine sarcoma viruses . similarly , enhanced replication of influenza virus and legionella pneumophila was observed in the lungs of nicotine - treated animals and cells lines , respectively . based on this observation , therefore , it could be suggested that reduced salivary immunoglobulin levels , especially igm , could play an important role in the pathogenesis of oral diseases in cigarette smokers and thus could have potential benefit in screening smokers at risk of developing oral diseases . although only salivary igm was significantly low in smokers , the observed non - significant reduction in all the classes of salivary immunoglobulin suggests pan - hypogammaglobulin in them . further study is required to provide explanation for the reported blood polyclonal b cells activation and the significantly reduced salivary igm levels observed in these cigarette smokers . small sample size ( due to strict inclusion criteria ) , unsuitable samples ( occasioned by stimulation or collected after 11 am ) , and exclusion of ziehl - neelson positive patients were some of the limitations of this study . this observation suggests that reduced salivary immunoglobulin level of igm might be involved in the pathogenesis of oral diseases in cigarette smokers .
background : cigarette smoking is a worldwide social epidemic and it is one of the main causes of preventable death and disability . gingivitis , periodontitis , pocket depth , attachment loss , alveolar bone loss , and tooth loss are some of oral pathologies commonly found in cigarette smokers . the aim of this study was to explore , for the first time among nigerians , the interplay between components of cigarette smoke and salivary levels of immunoglobulin classes so as to provide oral immunological based reasons for oral diseases in cigarette smokers.materials and methods : in this case - control study , 5 ml of unstimulated saliva was collected in plain sample bottles from 24 active smokers who smoke at least 6 sticks of cigarette per day and 21 sex and age - matched non - smokers who were apparently healthy . the samples were spun and supernatant stored at -20c until assayed . the immunoglobulin levels of the samples were estimated using enzyme - linked immunosorbent assay ( elisa ) . student 's t - test ( unpaired ) was used to determine significant differences between the two groups . p values less than 0.05 was considered significant.results:no significant differences were observed in the mean salivary levels of igg , iga , and ige . only igm was significantly lower in smokers compared with non - smokers ( p = 0.038 ) . the proportion of smokers with detectable level of salivary ige was lower compared with controls.conclusion:our study showed that there is decreased salivary igm in smokers . this observation suggests that reduced salivary immunoglobulin level of igm might be involved in the pathogenesis of oral diseases in cigarette smokers .